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Infections caused by Streptococcus pneumoniae (also known as pneumococci) pose a threat to human health. Pneumococcal infections are the most common cause of milder respiratory tract infections, such as otitis and sinusitis, and of more severe diseases, including pneumonia (with or without septicaemia) and meningitis. The introduction of pneumococcal conjugate vaccines in the childhood vaccination programme in many countries has led to a notable decrease of severe invasive pneumococcal disease in vaccinated children. However, infections caused by non-vaccine types have concurrently increased, causing invasive pneumococcal disease in unvaccinated populations (such as older adults), which has hampered the effect of these vaccines. Moreover, emerging antibiotic resistance is threatening effective therapy. Thus, new approaches are needed for the treatment and prevention of pneumococcal infections, and recent advances in the field may pave the way for new strategies. Recently, several important findings have been gained regarding pneumococcal epidemiology, genomics and the effect of the introduction of pneumococcal conjugate vaccines and of the COVID-19 pandemic. Moreover, elucidative pathogenesis studies have shown that the interactions between pneumococcal virulence factors and host receptors may be exploited for new therapies, and new vaccine candidates have been suggested. In this Review, we summarize some recent findings from clinical disease to basic pathogenesis studies that may be of importance for future control strategies., (© 2024. Springer Nature Limited.)

On September 20-22 September 2023, the international conference 'Microbiology 2023: from single cell to microbiome and host' convened microbiologists from across the globe for a very successful symposium, showcasing cutting-edge research in the field. Invited lecturers delivered exceptional presentations covering a wide range of topics, with a major emphasis on phages and microbiomes, on the relevant bacteria within these ecosystems, and their multifaceted roles in diverse environments. Discussions also spanned the intricate analysis of fundamental bacterial processes, such as cell division, stress resistance, and interactions with phages. Organized by four renowned Academies, the German Leopoldina, the French Académie des sciences, the Royal Society UK, and the Royal Swedish Academy of Sciences, the symposium provided a dynamic platform for experts to share insights and discoveries, leaving participants inspired and eager to integrate new knowledge into their respective projects. The success of Microbiology 2023 prompted the decision to host the next quadrennial academic meeting in Sweden. This choice underscores the commitment to fostering international collaboration and advancing the frontiers of microbiological knowledge. The transition to Sweden promises to be an exciting step in the ongoing global dialogue and specific collaborations on microbiology, a field where researchers will continue to push the boundaries of knowledge, understanding, and innovation not only in health and disease but also in ecology., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Staphylococcus aureus is a significant cause of morbidity and mortality in pulmonary infections. Patients with autosomal-dominant hyper-IgE syndrome due to STAT3 deficiency are particularly susceptible to acquiring staphylococcal pneumonia associated with lung tissue destruction. Because macrophages are involved in both pathogen defense and inflammation, we investigated the impact of murine myeloid STAT3 deficiency on the macrophage phenotype in vitro and on pathogen clearance and inflammation during murine staphylococcal pneumonia. Murine bone marrow-derived macrophages (BMDM) from STAT3 LysMCre+ knockout or Cre- wild-type littermate controls were challenged with S. aureus, LPS, IL-4, or vehicle control in vitro. Pro- and anti-inflammatory responses as well as polarization and activation markers were analyzed. Mice were infected intratracheally with S. aureus, bronchoalveolar lavage and lungs were harvested, and immunohistofluorescence was performed on lung sections. S. aureus infection of STAT3-deficient BMDM led to an increased proinflammatory cytokine release and to enhanced upregulation of costimulatory MHC class II and CD86. Murine myeloid STAT3 deficiency did not affect pathogen clearance in vitro or in vivo. Matrix metalloproteinase 9 was upregulated in Staphylococcus-treated STAT3-deficient BMDM and in lung tissues of STAT3 knockout mice infected with S. aureus. Moreover, the expression of miR-155 was increased. The enhanced inflammatory responses and upregulation of matrix metalloproteinase 9 and miR-155 expression in murine STAT3-deficient as compared with wild-type macrophages during S. aureus infections may contribute to tissue damage as observed in STAT3-deficient patients during staphylococcal pneumonia., (Copyright © 2023 The Authors.)

The bacterial cell envelope consists of multiple layers, including the peptidoglycan cell wall, one or two membranes, and often an external layer composed of capsular polysaccharides (CPS) or other components. How the synthesis of all these layers is precisely coordinated remains unclear. Here, we identify a mechanism that coordinates the synthesis of CPS and peptidoglycan in Streptococcus pneumoniae. We show that CPS synthesis initiates from the division septum and propagates along the long axis of the cell, organized by the tyrosine kinase system CpsCD. CpsC and the rest of the CPS synthesis complex are recruited to the septum by proteins associated with the divisome (a complex involved in septal peptidoglycan synthesis) but not the elongasome (involved in peripheral peptidoglycan synthesis). Assembly of the CPS complex starts with CpsCD, then CpsA and CpsH, the glycosyltransferases, and finally CpsJ. Remarkably, targeting CpsC to the cell pole is sufficient to reposition CPS synthesis, leading to diplococci that lack CPS at the septum. We propose that septal CPS synthesis is important for chain formation and complement evasion, thereby promoting bacterial survival inside the host., (© 2023. The Author(s).)

Pneumococcal conjugate vaccines (PCVs) used in childhood vaccination programs have resulted in replacement of vaccine-type with nonvaccine-type pneumococci in carriage and invasive pneumococcal disease (IPD). A vaccine based on highly conserved and protective pneumococcal antigens is urgently needed. Here, we performed intranasal immunization of mice with pneumococcal membrane particles (MPs) to mimic natural nasopharyngeal immunization. MP immunization gave excellent serotype-independent protection against IPD that was antibody dependent but independent of the cytotoxin pneumolysin. Using Western blotting, immunoprecipitation, mass spectrometry, and different bacterial mutants, we identified the conserved lipoproteins MalX and PrsA as the main antigens responsible for cross-protection. Additionally, we found that omitting the variable surface protein and vaccine candidate PspA from MPs enhanced protective immune responses to the conserved proteins. Our findings suggest that MPs containing MalX and PrsA could serve as a platform for pneumococcal vaccine development targeting the elderly and immunocompromised.

Increasing evidence highlights the role of bacteria in the physiopathology of cancer. However, the underlying molecular mechanisms remains poorly understood. Several cancer-associated bacteria have been shown to produce toxins which interfere with the host defense against tumorigenesis. Here, we show that lipopolysaccharides from Klebsiella pneumoniae and other Enterobacteria strongly inhibit the host tumor suppressor p53 pathway through a novel mechanism of p53 regulation. We found that lipopolysaccharides destabilize TP53 mRNA through a TLR4-NF-κB-mediated inhibition of the RNA-binding factor Wig-1. Importantly, we show that K. pneumoniae disables two major tumor barriers, oncogene-induced DNA damage signaling and senescence, by impairing p53 transcriptional activity upon DNA damage and oncogenic stress. Furthermore, we found an inverse correlation between the levels of TLR4 and p53 mutation in colorectal tumors. Hence, our data suggest that the repression of p53 by Enterobacteria via TLR4 alleviates the selection pressure for p53 oncogenic mutations and shapes the genomic evolution of cancer., (© 2022. The Author(s).)

Emerging antibiotic resistance demands identification of novel antibacterial compound classes. A bacterial whole-cell screen based on pneumococcal autolysin-mediated lysis induction was developed to identify potential bacterial cell wall synthesis inhibitors. A hit class comprising a 1-amino substituted tetrahydrocarbazole (THCz) scaffold, containing two essential amine groups, displayed bactericidal activity against a broad range of gram-positive and selected gram-negative pathogens in the low micromolar range. Mode of action studies revealed that THCz inhibit cell envelope synthesis by targeting undecaprenyl pyrophosphate-containing lipid intermediates and thus simultaneously inhibit peptidoglycan, teichoic acid, and polysaccharide capsule biosynthesis. Resistance did not readily develop in vitro, and the ease of synthesizing and modifying these small molecules, as compared to natural lipid II-binding antibiotics, makes THCz promising scaffolds for development of cell wall-targeting antimicrobials., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Streptococcus pneumoniae , a major cause of pneumonia, sepsis, and meningitis worldwide, has the nasopharynges of small children as its main ecological niche. Depletion of pneumococci from this niche would reduce the disease burden and could be achieved using small molecules with narrow-spectrum antibacterial activity. We identified the alkylated dicyclohexyl carboxylic acid 2CCA-1 as a potent inducer of autolysin-mediated lysis of S. pneumoniae , while having low activity against Staphylococcus aureus 2CCA-1-resistant strains were found to have inactivating mutations in fakB3 , known to be required for uptake of host polyunsaturated fatty acids, as well as through inactivation of the transcriptional regulator gene fabT , vital for endogenous, de novo fatty acid synthesis regulation. Structure activity relationship exploration revealed that, besides the central dicyclohexyl group, the fatty acid-like structural features of 2CCA-1 were essential for its activity. The lysis-inducing activity of 2CCA-1 was considerably more potent than that of free fatty acids and required growing bacteria, suggesting that 2CCA-1 needs to be metabolized to exert its antimicrobial activity. Total lipid analysis of 2CCA-1 treated bacteria identified unique masses that were modeled to 2CCA-1 containing lysophosphatidic and phosphatidic acid in wild-type but not in fakB3 mutant bacteria. This suggests that 2CCA-1 is metabolized as a fatty acid via FakB3 and utilized as a phospholipid building block, leading to accumulation of toxic phospholipid species. Analysis of FabT-mediated fakB3 expression elucidates how the pneumococcus could ensure membrane homeostasis and concurrent economic use of host-derived fatty acids. IMPORTANCE Fatty acid biosynthesis is an attractive antibiotic target, as it affects the supply of membrane phospholipid building blocks. In Streptococcus pneumoniae , it is not sufficient to target only the endogenous fatty acid synthesis machinery, as uptake of host fatty acids may bypass this inhibition. Here, we describe a small-molecule compound, 2CCA-1, with potent bactericidal activity that upon interactions with the fatty acid binding protein FakB3, which is present in a limited number of Gram-positive species, becomes metabolized and incorporated as a toxic phospholipid species. Resistance to 2CCA-1 developed specifically in fakB3 and the regulatory gene fabT These mutants reveal a regulatory connection between the extracellular polyunsaturated fatty acid metabolism and endogenous fatty acid synthesis in S. pneumoniae , which could ensure balance between efficient scavenging of host polyunsaturated fatty acids and membrane homeostasis. The data might be useful in the identification of narrow-spectrum treatment strategies to selectively target members of the Lactobacillales such as S. pneumoniae ., (Copyright © 2020 Reithuber et al.)

Influenza A virus (IAV)-related mortality is often due to secondary bacterial infections, primarily by pneumococci. Here, we study how IAV-modulated changes in the lungs affect bacterial replication in the lower respiratory tract (LRT). Bronchoalveolar lavages (BALs) from coinfected mice showed rapid bacterial proliferation 4 to 6 h after pneumococcal challenge. Metabolomic and quantitative proteomic analyses demonstrated capillary leakage with efflux of nutrients and antioxidants into the alveolar space. Pneumococcal adaptation to IAV-induced inflammation and redox imbalance increased the expression of the pneumococcal chaperone/protease HtrA. Presence of HtrA resulted in bacterial growth advantage in the IAV-infected LRT and protection from complement-mediated opsonophagocytosis due to capsular production. Absence of HtrA led to growth arrest in vitro that was partially restored by antioxidants. Pneumococcal ability to grow in the IAV-infected LRT depends on the nutrient-rich milieu with increased levels of antioxidants such as ascorbic acid and its ability to adapt to and cope with oxidative damage and immune clearance., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains. IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection., (Copyright © 2020 Vidaillac et al.)

The Gram-positive bacterium Streptococcus pneumoniae, the pneumococcus, is an important commensal resident of the human nasopharynx. Carriage is usually asymptomatic, however, S. pneumoniae can become invasive and spread from the upper respiratory tract to the lungs causing pneumonia, and to other organs to cause severe diseases such as bacteremia and meningitis. Several pneumococcal proteins important for its disease-causing capability have been described and many are expressed on the bacterial surface. The surface located pneumococcal type-1 pilus has been associated with virulence and the inflammatory response, and it is present in 20%-30% of clinical isolates. Its tip protein RrgA has been shown to be a major adhesin to human cells and to promote invasion through the blood-brain barrier. In this review we discuss recent findings of the impact of RrgA on bacterial colonization of the upper respiratory tract and on pneumococcal virulence, and use epidemiological data and genome-mining to suggest trade-off mechanisms potentially explaining the rather low prevalence of pilus-1 expressing pneumococci in humans., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Streptococcus pneumoniae (the pneumococcus) is a human respiratory tract pathogen and a major cause of morbidity and mortality globally. Although the pneumococcus is a commensal bacterium that colonizes the nasopharynx, it also causes lethal diseases such as meningitis, sepsis, and pneumonia, especially in immunocompromised patients, in the elderly, and in young children. Due to the acquisition of antibiotic resistance and the emergence of nonvaccine serotypes, the pneumococcus has been classified as one of the priority pathogens for which new antibacterials are urgently required by the World Health Organization, 2017. Understanding molecular mechanisms behind the pathogenesis of pneumococcal infections and bacterial interactions within the host is crucial to developing novel therapeutics. Previously considered to be an extracellular pathogen, it is becoming evident that pneumococci may also occasionally establish intracellular niches within the body to escape immune surveillance and spread within the host. Intracellular survival within host cells also enables pneumococci to resist many antibiotics. Within the host cell, the bacteria exist in unique vacuoles, thereby avoiding degradation by the acidic lysosomes, and modulate the expression of its virulence genes to adapt to the intracellular environment. To invade and survive intracellularly, the pneumococcus utilizes a combination of virulence factors such as pneumolysin (PLY), pneumococcal surface protein A (PspA), pneumococcal adhesion and virulence protein B (PavB), the pilus-1 adhesin RrgA, pyruvate oxidase (SpxB), and metalloprotease (ZmpB). In this review, we discuss recent findings showing the intracellular persistence of Streptococcus pneumoniae and its underlying mechanisms., (© 2019 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

Understanding the interplay between antibiotic resistance and bacterial fitness and virulence is essential to guide individual treatments and improve global antibiotic policies. A paradigmatic example of a resistance mechanism is the intrinsic inducible chromosomal β-lactamase AmpC from multiple Gram-negative bacteria, including Pseudomonas aeruginosa, a major nosocomial pathogen. The regulation of ampC expression is intimately linked to peptidoglycan recycling, and AmpC-mediated β-lactam resistance is frequently mediated by inactivating mutations in ampD, encoding an N-acetyl-anhydromuramyl-l-alanine amidase, affecting the levels of ampC-activating muropeptides. Here we dissect the impact of the multiple pathways causing AmpC hyperproduction on P. aeruginosa fitness and virulence. Through a detailed analysis, we demonstrate that the lack of all three P. aeruginosa AmpD amidases causes a dramatic effect in fitness and pathogenicity, severely compromising growth rates, motility, and cytotoxicity; the latter effect is likely achieved by repressing key virulence factors, such as protease LasA, phospholipase C, or type III secretion system components. We also show that ampC overexpression is required but not sufficient to confer the growth-motility-cytotoxicity impaired phenotype and that alternative pathways leading to similar levels of ampC hyperexpression and resistance, such as those involving PBP4, had no fitness-virulence cost. Further analysis indicated that fitness-virulence impairment is caused by overexpressing ampC in the absence of cell wall recycling, as reproduced by expressing ampC from a plasmid in an AmpG (muropeptide permease)-deficient background. Thus, our findings represent a major step in the understanding of β-lactam resistance biology and its interplay with fitness and pathogenesis., Importance: Understanding the impact of antibiotic resistance mechanisms on bacterial pathogenesis is critical to curb the spread of antibiotic resistance. A particularly noteworthy antibiotic resistance mechanism is the β-lactamase AmpC, produced by Pseudomonas aeruginosa, a major pathogen causing hospital-acquired infections. The regulation of AmpC is linked to the cell wall recycling pathways, and frequently, resistance to β-lactams is caused by mutation of several of the components of the cell wall recycling pathways such as AmpD. Here we dissect the impact of the pathways for AmpC hyperproduction on virulence, showing that the lack of all three P. aeruginosa AmpD amidases causes a major effect in fitness and pathogenicity, compromising growth, motility, and cytotoxicity. Further analysis indicated that fitness-virulence impairment is specifically caused by the hyperproduction of AmpC in the absence of cell wall recycling. Our work provides valuable information for delineating future strategies for combating P. aeruginosa infections by simultaneously targeting virulence and antibiotic resistance., (Copyright © 2016 Pérez-Gallego et al.)

Virulent group A streptococci have become a serious threat, with the emergence of the hypervirulent lineage M1T1. In this issue of Cell Host & Microbe, LaRock et al. (2015), uncover a role for the streptococcal M1 protein in neutralizing a key human antimicrobial peptide, cathelicidin., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Transport of DNA across bacterial membranes involves complex DNA uptake systems. In Gram-positive bacteria, the DNA uptake machinery shares fundamental similarities with type IV pili and type II secretion systems. Although dedicated pilus structures, such as type IV pili in Gram-negative bacteria, are necessary for efficient DNA uptake, the role of similar structures in Gram-positive bacteria is just beginning to emerge. Recently two essentially very different pilus structures composed of the same major pilin protein ComGC were proposed to be involved in transformation of the Gram-positive bacterium Streptococcus pneumoniae - one is a long, thin, type IV pilus-like fiber with DNA binding capacity and the other one is a pilus structure that was thicker, much shorter and not able to bind DNA. Here we discuss how competence induced pili, either by pilus retraction or by a transient pilus-related opening in the cell wall, may mediate DNA uptake in S. pneumoniae., (© 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.)

Background: Streptococcus pneumoniae serotype 1 has a high likelihood of causing invasive disease. Serotype 1 isolates belonging to CC228 are associated with low mortality, while CC217 isolates exhibit high mortality in patients., Methods: Clinical pneumococcal isolates and mutants were evaluated in wild-type C57BL/6 mice, macrophage-depleted mice, neutrophil-depleted mice, and SIGN-R1 knockout mice. In vitro models included binding and phagocytosis by THP-1 cells, capsule measurements, hydrogen peroxide production, and viability assays., Results: During early systemic infection in mice with serotype 1, large-colony variants appeared in blood. Similar large colonies were found in blood specimens from patients with invasive disease. Large morphotypes contained higher numbers of viable bacteria, grew faster, produced no or little hydrogen peroxide, and contained mutations in the spxB gene. spxB mutants were considerably more virulent in wild-type mice, less susceptible to early host clearance than wild-type strains after intravenous infection, but impaired in colonization. spxB mutants were less efficiently phagocytosed by macrophages than wild-type bacteria, which, in contrast to spxB mutants, caused more-severe disease when macrophages or SIGN-R1 were depleted., Conclusions: Hypervirulent spxB mutants are selected in both mice and patients and are resistant to early macrophage-mediated clearance.

Spread of antibiotic resistance is mediated by clonal lineages of bacteria that besides being resistant also possess other properties promoting their success. Some vaccines already in use, such as the pneumococcal conjugate vaccines, have had an effect on these successful clones, but at the same time have allowed for the expansion and resistance evolution of previously minor clones not covered by the vaccine. Since resistance frequently is horizontally transferred it will be difficult to generate a vaccine that covers all possible genetic lineages prone to develop resistance unless the vaccine target(s) is absolutely necessary for spread and/or disease development. Targeting the resistance mechanism itself by a vaccine is an interesting but hitherto unexplored approach.

Streptococcus pneumoniae is a major human pathogen that successfully adapts to the host environment via an efficient uptake system for free DNA liberated from other organisms in the upper respiratory tract, facilitating immune evasion and drug resistance. Although the initial signaling events leading to pneumococcal competence for DNA transformation and the fate of DNA when it has been taken up have been extensively studied, the actual mechanism by which DNA in the environment may traverse the thick capsular and cell wall layers remains unknown. Here we visualize that induction of competence results in the formation of a native morphologically distinct pilus structure on the bacterial surface. This plaited pilus is encoded by the competence (com)G locus, and, after assembly, it is rapidly released into the surrounding medium. Heterologous pneumococcal pilus expression in Escherichia coli was obtained by replacing the pulE-K putative pilin genes of the Klebsiella oxytoca type II secretion system with the complete comG locus. In the pneumococcus, the coordinated secretion of pili from the cells correlates to DNA transformation. A model for DNA transformation is proposed whereby pilus assembly "drills" a channel across the thick cell wall that becomes transiently open by secretion of the pilus, providing the entry port for exogenous DNA to gain access to DNA receptors associated with the cytoplasmic membrane.

Unlabelled: The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs., Importance: Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

Background: Pneumococcal serotypes are represented by a varying number of clonal lineages with different genetic contents, potentially affecting invasiveness. However, genetic variation within the same genetic lineage may be larger than anticipated., Methods: A total of 715 invasive and carriage isolates from children in the same region and during the same period were compared using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Bacterial genome sequencing, functional assays, and in vivo virulence mice studies were performed., Results: Clonal types of the same serotype but also intraclonal variants within clonal complexes (CCs) showed differences in invasive-disease potential. CC138, a common CC, was divided into several PFGE patterns, partly explained by number, location, and type of temperate bacteriophages. Whole-genome sequencing of 4 CC138 isolates representing PFGE clones with different invasive-disease potentials revealed intraclonal sequence variations of the virulence-associated proteins pneumococcal surface protein A (PspA) and pneumococcal choline-binding protein C (PspC). A carrier isolate lacking PcpA exhibited decreased virulence in mice, and there was a differential binding of human factor H, depending on invasiveness., Conclusions: Pneumococcal clonal types but also intraclonal variants exhibited different invasive-disease potentials in children. Intraclonal variants, reflecting different prophage contents, showed differences in major surface antigens. This suggests ongoing immune selection, such as that due to PspC-mediated complement resistance through varied human factor H binding, that may affect invasiveness in children.

Streptococcus pneumoniae is a major human pathogen, and a leading cause of disease and death worldwide. Pneumococcal invasive disease is triggered by initial asymptomatic colonization of the human upper respiratory tract. The pneumococcal serine-rich repeat protein (PsrP) is a lung-specific virulence factor whose functional binding region (BR) binds to keratin-10 (KRT10) and promotes pneumococcal biofilm formation through self-oligomerization. We present the crystal structure of the KRT10-binding domain of PsrP (BR187-385) determined to 2.0 Å resolution. BR187-385 adopts a novel variant of the DEv-IgG fold, typical for microbial surface components recognizing adhesive matrix molecules adhesins, despite very low sequence identity. An extended β-sheet on one side of the compressed, two-sided barrel presents a basic groove that possibly binds to the acidic helical rod domain of KRT10. Our study also demonstrates the importance of the other side of the barrel, formed by extensive well-ordered loops and stabilized by short β-strands, for interaction with KRT10.

Virulence factor secretion and assembly occurs at spatially restricted foci in some Gram-positive bacteria. Given the essentiality of the general secretion pathway in bacteria and the contribution of virulence factors to disease progression, the foci that coordinate these processes are attractive antimicrobial targets. In this study, we show in Enterococcus faecalis that SecA and Sortase A, required for the attachment of virulence factors to the cell wall, localize to discrete domains near the septum or nascent septal site as the bacteria proceed through the cell cycle. We also demonstrate that cationic human β-defensins interact with E. faecalis at discrete septal foci, and this exposure disrupts sites of localized secretion and sorting. Modification of anionic lipids by multiple peptide resistance factor, a protein that confers antimicrobial peptide resistance by electrostatic repulsion, renders E. faecalis more resistant to killing by defensins and less susceptible to focal targeting by the cationic antimicrobial peptides. These data suggest a paradigm in which focal targeting by antimicrobial peptides is linked to their killing efficiency and to disruption of virulence factor assembly.

Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.

Secondary infections with Streptococcus pneumoniae (SP) are frequently observed following influenza A virus (IAV) infection and have a substantial impact on global health. Despite this, the basis for the disease progression is incompletely understood. To investigate the effect of co-infection on human monocyte-derived dendritic cells (MDDCs) we analysed the expression of clinically important pro-inflammatory and immune-modulatory cytokines. IAV infection or treatment with supernatants from IAV-infected cell cultures resulted in priming of the DCs which subsequently influenced the production of IL-12p70, as well as IL-6, following SP infection. Co-infection of the same cell was not required but this effect was dependent on the time, dose and duration of the infections, as well as pathogen viability, bacterial uptake and endosome acidification. Bacterially infected cells were characterized as the main producers of IL-12p70. Finally, we showed that type I interferons were primarily responsible for the priming of IL-12p70 that was observed by infection with IAV. These results provide a probable mechanism for the elevated levels of particular cytokines observed in IAV and SP co-infected cell cultures with implications for the pathogenic outcome observed during in vivo infection., (© 2013 John Wiley & Sons Ltd.)

Streptococcus pneumoniae of serotype 3 possess a mucoid capsule and cause disease associated with high mortality rates relative to other pneumococci. Phylogenetic analysis of a complete reference genome and 81 draft sequences from clonal complex 180, the predominant serotype 3 clone in much of the world, found most sampled isolates belonged to a clade affected by few diversifying recombinations. However, other isolates indicate significant genetic variation has accumulated over the clonal complex's entire history. Two closely related genomes, one from the blood and another from the cerebrospinal fluid, were obtained from a patient with meningitis. The pair differed in their behaviour in a mouse model of disease and in their susceptibility to antimicrobials, with at least some of these changes attributable to a mutation that up-regulated the patAB efflux pump. This indicates clinically important phenotypic variation can accumulate rapidly through small alterations to the genotype., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: Pneumococcal pili have been shown to influence pneumococcal colonization, disease development, and the inflammatory response in mice. The role of the pilus-associated RrgA adhesin in pneumococcal interactions with murine and human macrophages was investigated. Expression of pili with RrgA enhanced the uptake of pneumococci by murine and human macrophages that was abolished by antibodies to complement receptor 3 (CR3) and not seen in CR3-deficient macrophages. Recombinant RrgA, but not pilus subunit RrgC, promoted CR3-mediated phagocytosis of coated beads by murine and human macrophages. Flow cytometry showed that purified CR3 binds pneumococcal cells expressing RrgA, and purified RrgA was shown to interact with CR3 and its I domain. In vivo, RrgA facilitated spread of pneumococci from the upper airways and peritoneal cavity to the bloodstream. Earlier onset of septicemia and more rapidly progressing disease was observed in wild-type mice compared to CR3-deficient mice challenged intranasally or intraperitoneally with pneumococci. Motility assays and time-lapse video microscopy showed that pneumococcal stimulation of macrophage motility required RrgA and CR3. These findings, together with the observed RrgA-dependent increase of intracellular survivors up to 10 h following macrophage infection, suggest that RrgA-CR3-mediated phagocytosis promotes systemic pneumococcal spread from local sites., Importance: Streptococcus pneumoniae is a major contributor to morbidity and mortality in infectious diseases globally. Symptomatology is mainly due to pneumococcal interactions with host cells leading to an inflammatory response. However, we still need more knowledge on how pneumococci talk to immune cells and the importance of this interaction. Recently, a novel structure was identified on the pneumococcal surface, an adhesive pilus found in about 30% of clinical pneumococcal isolates. The pilus has been suggested to be important for successful spread of antibiotic-resistant pneumococcal clones globally. Here we sought to identify mechanisms for how the pneumococcal pilin subunit RrgA contributes to disease development by interacting with host immune cells. Our data suggest a new way for how pneumococci may cross talk with phagocytic cells and affect disease progression. An increased understanding of these processes may lead to better strategies for how to treat these common infections.

Unlabelled: Though the bacterial opportunist Enterococcus faecalis causes a myriad of hospital-acquired infections (HAIs), including catheter-associated urinary tract infections (CAUTIs), little is known about the virulence mechanisms that it employs. However, the endocarditis- and biofilm-associated pilus (Ebp), a member of the sortase-assembled pilus family, was shown to play a role in a mouse model of E. faecalis ascending UTI. The Ebp pilus comprises the major EbpC shaft subunit and the EbpA and EbpB minor subunits. We investigated the biogenesis and function of Ebp pili in an experimental model of CAUTI using a panel of chromosomal pilin deletion mutants. A nonpiliated pilus knockout mutant (EbpABC(-) strain) was severely attenuated compared to its isogenic parent OG1RF in experimental CAUTI. In contrast, a nonpiliated ebpC deletion mutant (EbpC(-) strain) behaved similarly to OG1RF in vivo because it expressed EbpA and EbpB. Deletion of the minor pilin gene ebpA or ebpB perturbed pilus biogenesis and led to defects in experimental CAUTI. We discovered that the function of Ebp pili in vivo depended on a predicted metal ion-dependent adhesion site (MIDAS) motif in EbpA's von Willebrand factor A domain, a common protein domain among the tip subunits of sortase-assembled pili. Thus, this study identified the Ebp pilus as a virulence factor in E. faecalis CAUTI and also defined the molecular basis of this function, critical knowledge for the rational development of targeted therapeutics., Importance: Catheter-associated urinary tract infections (CAUTIs), one of the most common hospital-acquired infections (HAIs), present considerable treatment challenges for physicians. Inherently resistant to several classes of antibiotics and with a propensity to acquire vancomycin resistance, enterococci are particularly worrisome etiologic agents of CAUTI. A detailed understanding of the molecular basis of Enterococcus faecalis pathogenesis in CAUTI is necessary for the development of preventative and therapeutic strategies. Our results elucidated the importance of the E. faecalis Ebp pilus and its subunits for enterococcal virulence in a mouse model of CAUTI. We further showed that the metal ion-dependent adhesion site (MIDAS) motif in EbpA is necessary for Ebp function in vivo. As this motif occurs in other sortase-assembled pili, our results have implications for the molecular basis of virulence not only in E. faecalis CAUTI but also in additional infections caused by enterococci and other Gram-positive pathogens.

The pneumococcal autolysin LytA is a virulence factor involved in autolysis as well as in fratricidal- and penicillin-induced lysis. In this study, we used biochemical and molecular biological approaches to elucidate which factors control the cytoplasmic translocation and lytic activation of LytA. We show that LytA is mainly localized intracellularly, as only a small fraction was found attached to the extracellular cell wall. By manipulating the extracellular concentration of LytA, we found that the cells were protected from lysis during exponential growth, but not in the stationary phase, and that a defined threshold concentration of extracellular LytA dictates the onset of autolysis. Stalling growth through nutrient depletion, or the specific arrest of cell wall synthesis, sensitized cells for LytA-mediated lysis. Inhibition of cell wall association via the choline binding domain of an exogenously added enzymatically inactive form of LytA revealed a potential substrate for the amidase domain within the cell wall where the formation of nascent peptidoglycan occurs.

Chlamydiae are Gram-negative, obligate intracellular pathogens that replicate within a membrane-bounded compartment termed an inclusion. Throughout their development, they actively modify the eukaryotic environment. The type III secretion (TTS) system is the main process by which the bacteria translocate effector proteins into the inclusion membrane and the host cell cytoplasm. Here we describe a family of type III secreted effectors that are present in all pathogenic chlamydiae and absent in the environment-related species. It is defined by a common domain of unknown function, DUF582, that is present in four or five proteins in each Chlamydiaceae species. We show that the amino-terminal extremity of DUF582 proteins functions as a TTS signal. DUF582 proteins from C. trachomatis CT620, CT621, and CT711 are expressed at the middle and late phases of the infectious cycle. Immunolocalization further revealed that CT620 and CT621 are secreted into the host cell cytoplasm, as well as within the lumen of the inclusion, where they do not associate with bacterial markers. Finally, we show that DUF582 proteins are present in nuclei of infected cells, suggesting that members of the DUF582 family of effector proteins may target nuclear cell functions. The expansion of this family of proteins in pathogenic chlamydiae and their conservation among the different species suggest that they play important roles in the infectious cycle.

Background: Patients with primary immunodeficiency (PID) often suffer from frequent respiratory tract infections. Despite standard treatment with IgG-substitution and antibiotics many patients do not improve significantly. Therefore, we hypothesized that additional immune deficits may be present among these patients., Objective: To investigate if PID patients exhibit impaired production of antimicrobial peptides (AMPs) in nasal fluid and a possible link between AMP-expression and Th17-cells., Methods: Nasal fluid, nasopharyngeal swabs and peripheral blood mononuclear cells (PBMCs) were collected from patients and healthy controls. AMP levels were measured in nasal fluid by Western blotting. Nasal swabs were cultured for bacteria. PBMCs were stimulated with antigen and the supernatants were assessed for IL-17A release by ELISA., Results: In healthy controls and most patients, AMP levels in nasal fluid were increased in response to pathogenic bacteria. However, this increase was absent in patients with common variable immunodeficiency (CVID) and Hyper-IgE syndrome (HIES), despite the presence of pathogenic bacteria. Furthermore, stimulation of PBMCs revealed that both HIES and CVID patients exhibited an impaired production of IL-17A., Conclusion: CVID and HIES patients appear to have a dysregulated AMP response to pathogenic bacteria in the upper respiratory tract, which could be linked to an aberrant Th17 cell response., (© 2011 Cederlund et al.)

The human microbiota has come into focus in the search for component causes of chronic diseases, such as gastrointestinal cancers. Presumably long induction periods and altered local environments after disease onset call for the development of methods for characterization of microorganisms colonizing the host decades before disease onset. Sequencing of microbial genomes in old formalin-fixed and paraffin-embedded (FFPE) gastrointestinal biopsies provides a means for such studies but is still challenging. Here we report a method based on laser capture micro-dissection and modified Roche 454 high-throughput pyrosequencing to obtain metagenomic profiles of Helicobacter pylori. We applied this method to two 15 year old FFPE biopsies from two patients. Frozen homogenized biopsies from the same gastroscopy sessions were also available for comparison after re-culture of H. pylori. For both patients, H. pylori DNA dissected from FFPE sections had ~96.4% identity with culture DNA from the same patients, while only ~92.5% identity with GenBank reference genomes, and with culture DNA from the other patient. About 82% and 60% of the predicted genes in the two genomes were captured by at least a single sequencing read. Along with sequences displaying high similarity to known H. pylori genes, novel and highly variant H. pylori sequences were identified in the FFPE sections by our physical enrichment approach, which would likely not have been detected by a sequence capture approach. The study demonstrates the feasibility of longitudinal metagenomic studies of H. pylori using decade-preserved FFPE biopsies.

Background: Statin treatment has been associated with a beneficial outcome on respiratory tract infections. In addition, previous in vitro and in vivo experiments have indicated favorable effects of statins in bacterial infections., Aim: The aim of the present study was to elucidate possible antibacterial effects of statins against primary pathogens of the respiratory tract., Methods: MIC-values for simvastatin, fluvastatin and pravastatin against S. pneumoniae, M. catarrhalis and H. influenzae were determined by traditional antibacterial assays. A BioScreen instrument was used to monitor effects of statins on bacterial growth and to assess possible synergistic effects with penicillin. Bacterial growth in whole blood and serum from healthy volunteers before and after a single dose of simvastatin, fluvastatin and penicillin (positive control) was determined using a blood culture system (BactAlert)., Findings: The MIC-value for simvastatin against S pneumoniae and M catarrhalis was 15 µg/mL (36 mmol/L). Fluvastatin and Pravastatin showed no antibacterial effect in concentrations up to 100 µg/mL (230 µmol/L). Statins did not affect growth or viability of H influenzae. Single doses of statins given to healthy volunteers did not affect growth of pneumococci, whereas penicillin efficiently killed all bacteria., Conclusions: Simvastatin at high concentrations 15 µg/mL (36 µmol/L) rapidly kills S pneumoniae and M catarrhalis. However, these concentrations by far exceed the concentrations detected in human blood during simvastatin therapy (1-15 nmol/L) and single doses of statins given to healthy volunteers did not improve antibacterial effects of whole blood. Thus, a direct bactericidal effect of statins in vivo is probably not the mechanism behind the observed beneficial effect of statins against various infections.

Many important pathogens have humans as their normal ecological niche where healthy carriage dominates over disease. The ability of these commensal pathogens, such as Streptococcus pneumoniae, to cause disease depends on a series of microbial factors as well as of genetic and environmental factors in the human host affecting the clearing capacity mediated by the innate and adaptive immune system. This delicate interplay between microbe and host affects not only the likelihood for a commensal pathogen to cause disease, but also disease type and disease severity., (Copyright 2010 Elsevier Inc. All rights reserved.)

Most secretion pathways in bacteria and eukaryotic cells are challenged by the requirement for their substrate proteins to mature after they traverse a membrane barrier and enter a reactive oxidizing environment. For Gram-positive bacteria, the mechanisms that protect their exported proteins from misoxidation during their post-translocation maturation are poorly understood. To address this, we separated numerous bacterial species according to their tolerance for oxygen and divided their proteomes based on the predicted subcellular localization of their proteins. We then applied a previously established computational approach that utilizes cysteine incorporation patterns in proteins as an indicator of enzymatic systems that may exist in each species. The Sec-dependent exported proteins from aerobic Gram-positive Actinobacteria were found to encode cysteines in an even-biased pattern indicative of a functional disulfide bond formation system. In contrast, aerobic Gram-positive Firmicutes favor the exclusion of cysteines from both their cytoplasmic proteins and their substantially longer exported proteins. Supporting these findings, we show that Firmicutes, but not Actinobacteria, tolerate growth in reductant. We further demonstrate that the actinobacterium Corynebacterium glutamicum possesses disulfide-bonded proteins and two dimeric Dsb-like enzymes that can efficiently catalyze the formation of disulfide bonds. Our results suggest that cysteine exclusion is an important adaptive strategy against the challenges presented by oxidative environments.

The respiratory tract pathogen Streptococcus pneumoniae is a primary cause of morbidity and mortality worldwide. Pili enhance initial adhesion as well as the capacity of pneumococci to cause pneumonia and bacteremia. Pilus-associated sortases (SrtB, SrtC, and SrtD) are involved in the biogenesis of pneumococcal pili, composed of repeating units of RrgB that create the stalk to which the RrgA adhesin and the preferential pilus tip subunit RrgC are covalently associated. Using single sortase-expressing strains, we demonstrate that both pilin-polymerizing sortases SrtB and SrtC can covalently link pili to the peptidoglycan cell wall, a property shared with the non-pilus-polymerizing enzyme SrtD and the housekeeping sortase SrtA. Comparative analysis of the crystal structures of S. pneumoniae SrtC and SrtB revealed structural differences explaining the incapacity of SrtC, but not of SrtB, to incorporate RrgC into the pilus. Accordingly, site-directed mutagenesis of Thr(160) in SrtB to an arginine as in SrtC (Arg(160)) partially converted its substrate specificity into that of SrtC. Solving two crystal structures for SrtC suggests that an opening of a flexible lid and a concomitant cysteine rotation are important for catalysis and the activation of the catalytic cysteine of pilus-associated sortases.

Dendritic cells (DCs) protect the respiratory epithelium via induction of innate immune responses and priming of naïve T cells during the initiation of adaptive immunity. Streptococcus pneumoniae, a commonly carried asymptomatic member of the human nasopharyngeal microflora, can cause invasive and inflammatory diseases and the cholesterol-dependent cytotoxin pneumolysin is a major pneumococcal virulence factor implicated in compounding tissue damage and mediating inflammatory responses. While most studies examining the impact of pneumolysin have been based on murine models, we have focused this study on human DC responses. We show that expression of haemolytic pneumolysin inhibits human DC maturation, induction of proinflammatory cytokines and activation of the inflammasome. Furthermore, intracellular production of pneumolysin induces caspase-dependent apoptosis in infected DCs. Similarly, clinical isolates with non-haemolytic pneumolysin were more proinflammatory and caused less apoptosis compared to clonally related strains with active pneumolysin. This study describes a novel role of pneumolysin in the evasion of human DC surveillance that could have a profound clinical impact upon inflammatory disease progression and highlights the need to study human responses to human-specific pathogens.

Most commensal and pathogenic bacteria interacting with eukaryotic hosts express adhesive molecules on their surfaces that promote interaction with host cell receptors or with soluble macromolecules. Even though bacterial attachment to epithelial cells may be beneficial for bacterial colonization, adhesion may come at a cost because bacterial attachment to immune cells can facilitate phagocytosis and clearing. Many pathogenic bacteria have solved this dilemma by producing an antiphagocytic surface layer usually consisting of polysaccharide and by expressing their adhesins on polymeric structures that extend out from the cell surface. In this review, we will focus on the interaction between bacterial adhesins and the host, with an emphasis on pilus-like structures.

The human gastric pathogen Helicobacter pylori colonizes the stomachs of half of the human population, and causes development of peptic ulcer disease and gastric adenocarcinoma. H. pylori-associated chronic atrophic gastritis (ChAG) with loss of the acid-producing parietal cells, is correlated with an increased risk for development of gastric adenocarcinoma. The majority of H. pylori isolates produce lipopolysaccharides (LPS) decorated with human-related Lewis epitopes, which have been shown to phase-vary in response to different environmental conditions. We have characterized the adaptations of H. pylori LPS and Lewis antigen expression to varying gastric conditions; in H. pylori isolates from mice with low or high gastric pH, respectively; in 482 clinical isolates from healthy individuals and from individuals with ChAG obtained at two time points with a four-year interval between endoscopies; and finally in isolates grown at different pH in vitro. Here we show that the gastric environment can contribute to a switch in Lewis phenotype in the two experimental mouse models. The clinical isolates from different human individuals showed that intra-individual isolates varied in Lewis antigen expression although the LPS diversity was relatively stable within each individual over time. Moreover, the isolates demonstrated considerable diversity in the levels of glycosylation and in the sizes of fucosylated O-antigen chains both within and between individuals. Thus our data suggest that different LPS variants exist in the colonizing H. pylori population, which can adapt to changes in the gastric environment and provide a means to regulate the inflammatory response of the host during disease progression.

Marine bacteria can cause harm to single-celled and multicellular eukaryotes. However, relatively little is known about the underlying genetic basis for marine bacterial interactions with higher organisms. We examined whole-genome sequences from a large number of marine bacteria for the prevalence of homologues to virulence genes and pathogenicity islands known from bacteria that are pathogenic to terrestrial animals and plants. As many as 60 out of 119 genomes of marine bacteria, with no known association to infectious disease, harboured genes of virulence-associated types III, IV, V and VI protein secretion systems. Type III secretion was relatively uncommon, while type IV was widespread among alphaproteobacteria (particularly among roseobacters) and type VI was primarily found among gammaproteobacteria. Other examples included homologues of the Yersinia murine toxin and a phage-related 'antifeeding' island. Analysis of the Global Ocean Sampling metagenomic data indicated that virulence genes were present in up to 8% of the planktonic bacteria, with highest values in productive waters. From a marine ecology perspective, expression of these widely distributed genes would indicate that some bacteria infect or even consume live cells, that is, generate a previously unrecognized flow of organic matter and nutrients directly from eukaryotes to bacteria.

Background: Chlamydiae are obligate intracellular pathogens that possess a type III secretion system to deliver proteins into the host cell during infection. Small molecule inhibitors of type III secretion in Yersinia, termed INPs (Innate Pharmaceuticals AB) were reported to strongly inhibit Chlamydia growth in epithelial cells. In this study we have analyzed the effect of these drugs on bacterial invasiveness., Results: We demonstrate that INPs affect Chlamydia growth in a dose dependent manner after bacterial invasion. The efficiency of C. trachomatis L2 and C. caviae GPIC entry into host cells was not altered in the presence of INPs. In C. caviae, entry appears to proceed normally with recruitment of actin and the small GTPases Rac, Cdc42 and Arf6 to the site of bacterial entry., Conclusion: INPs have a strong inhibitory effect on Chlamydia growth. However, bacterial invasion is not altered in the presence of these drugs. In the light of these results, we discuss several hypotheses regarding the mode of action of INPs on type III secretion during the Chlamydia infectious cycle.

The rlrA genetic islet encodes an extracellular pilus in the Gram-positive pathogen Streptococcus pneumoniae. Of the three genes for structural subunits, rrgB encodes the major pilin, while rrgA and rrgC encode ancillary pilin subunits decorating the pilus shaft and tip. Deletion of all three pilus-associated sortase genes, srtB, srtC and srtD, completely prevents pilus biogenesis. Expression of srtB alone is sufficient to covalently associate RrgB subunits to one another as well as linking the RrgA adhesin and the RrgC subunit into the polymer. The active-site cysteine residue of SrtB (Cys 177) is crucial for incorporating RrgC, even when the two other sortase genes are expressed. SrtC is redundant to SrtB in permitting RrgB polymerization, and in linking RrgA to the RrgB filament, but SrtC is insufficient to incorporate RrgC. In contrast, expression of srtD alone fails to mediate RrgB polymerization, and a srtD mutant assembles heterotrimeric pilus indistinguishable from wild type. Topological studies demonstrate that pilus antigens are localized to symmetric foci at the cell surface in the presence of all three sortases. This symmetric focal presentation is abrogated in the absence of either srtB or srtD, while deletion of srtC had no effect. In addition, strains expressing srtB alone or srtC alone also displayed disrupted antigen localization, despite polymerizing subunits. Our data suggest that both SrtB and SrtC act as pilus subunit polymerases, with SrtB processing all three pilus subunit proteins, while SrtC only RrgB and RrgA. In contrast, SrtD does not act as a pilus subunit polymerase, but instead is required for wild-type focal presentation of the pilus at the cell surface.

Globally spreading bacterial strains belong to clonal types that have the capacity to colonize, spread and cause disease in the community. Recent comparative genomic analyses of well-defined clinical isolates have led to the identification of bacterial properties that are required for the successful spread of bacterial clones. In this Review, we discuss the evolution of bacterial clones, the importance of recombination versus mutations for evolution of clones, common methods used to study clonal relationships among bacteria, factors that may contribute to the clonal spread of bacteria and the potential relevance of bacterial clones to clinical disease. We focus on the common pathogen Streptococcus pneumoniae, although other bacteria are also briefly discussed, such as Helicobacter pylori, Staphylococcus aureus and Mycobacterium tuberculosis.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Its polysaccharide capsule causes resistance to phagocytosis and interferes with the innate immune system's ability to clear infections at an early stage. Nevertheless, we found that encapsulated pneumococci are sensitive to killing by a human neutrophil granule extract. We fractionated the extract by high-performance liquid chromatography and identified alpha-defensins by mass spectrometry as the proteins responsible for killing pneumococci. Analysis of sensitivity to the commercial alpha-defensins human neutrophil proteins 1 to 3 (HNP1-3) confirmed these findings. We analyzed the sensitivities of different pneumococcal strains to HNP1-3 and found that encapsulated strains are efficiently killed at physiological concentrations (7.5 microg/ml). Surprisingly, nonencapsulated, nonvirulent pneumococci were significantly less sensitive to alpha-defensins. The proposed mechanisms of alpha-defensin resistance in nonencapsulated pneumococci is surface charge modification, e.g., by introduction of positive charge by D-alanylation of surface-exposed lipoteichoic acids. These mechanisms are surmounted by the presence of the capsule, which we hypothesize is masking these charge modifications. Hence, alpha-defensins in the phagolysosome of neutrophils possibly contribute to intracellular killing after antibody-mediated opsonophagocytosis of encapsulated pneumococci.

The lungs are continuously exposed to a broad array of microbes through inhalation, and microorganisms that escape clearance by the upper airway mucociliary motion will deposit in the alveolar compartment of the lower airways. The pulmonary epithelium in the alveolar compartment is covered by a thin aqueous layer that contains surfactant proteins but also microbicidal components. We have here identified the epithelial cell surface-expressed heparin/heparan sulfate interacting protein (HIP/RPL29) by high-performance liquid chromatography-fractionation, N-terminal sequencing, and mass spectrometry analysis as a major antimicrobial component in extracts of mouse lung tissue. HIP/RPL29 was also detected in extracts of mouse small intestinal tissue. HIP/RPL29 exhibited broad antibacterial activity, notably against Pseudomonas aeruginosa strains. Human recombinant HIP/RPL29 exhibited killing activity in the same order of magnitude. The HIP/RPL29 protein was demonstrated to be localized to the epithelial cells and cell surface of the lungs and intestines by immunohistochemistry. We suggest that HIP/RPL29 fulfills a function as an abundant antibacterial factor of the epithelial innate defense shield against invading bacteria in both the lungs and the small intestine.

Purpose of Review: The present review summarizes exciting new findings and reports recent advances in the understanding of the role of toll-like receptors in health and disease. It intends to provide a rough survey on topics discussed by researchers in the field and to stimulate discussion on new aspects of the complex processes involved in innate host defence., Recent Findings: Novel findings have been reported on the many aspects of toll-like receptors biology, namely the receptor structure and the molecular process of ligand recognition, receptor assembly, cellular localization and trafficking, downstream signaling and the regulatory factors involved, genetic polymorphisms within receptor genes and their linkage to human diseases, and the functional role of toll-like receptors in immune defence and host-microbe homeostasis., Summary: Recent advances have allowed a more detailed picture not only of the processes involved in microbial recognition and host defence but also revealed unexpected insights into the cause of inflammatory processes and the close interrelationship between the vertebrate host and the microbially colonized environment.

The chaperone/usher pathway is responsible for the assembly of adhesive pili on the surface of gram-negative pathogenic bacteria. In this issue, Remaut et al. (2008) present the crystal structure of the PapC usher translocation domain and images of the FimD usher bound to a pilus translocation intermediate. These new structures provide the first detailed view of a translocase in action.

Background: Streptococcus pneumoniae is a genetically diverse major human pathogen, yet a common colonizer of the nasopharynx. Here we analyzed the influence of defects affecting in vitro growth rate, on the ability of S. pneumoniae to colonize and to cause invasive disease in vivo., Results: Of eleven different clinical isolates one serotype 14 carrier isolate showed a significantly longer generation time as compared to other isolates, and was severely attenuated in mice. To directly investigate the impact of growth rate on virulence, a panel of mutants in five non-essential housekeeping genes was constructed in the virulent TIGR4 background by insertion-deletion mutagenesis. Three of these mutants (ychF, hemK and yebC) were, to different degrees, growth defective, and showed a reduced invasiveness in an intranasal murine challenge model that correlated to their in vitro growth rate, but remained capable of colonizing the upper airways. The growth defect, as well as virulence defect of the hemK insertion-deletion mutant, was mediated by polarity effects on the downstream yrdC gene, encoding a probable chaperone in ribosome assembly., Conclusion: We conclude that large fitness defects are needed to completely prevent pneumococci from causing invasive disease after intranasal challenge. However, even severe growth defects still allow pneumococci to persistently colonize the upper airways.