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All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by relieving bacterial stress and eliminating toxic compounds, as well as by facilitating membrane remodeling and ameliorating inhospitable environments. However, vesicle production comes with a price. It is energetically costly and, in the case of colonizing pathogens, it elicits host immune responses, which reduce bacterial viability. This raises an interesting paradox regarding why bacteria produce vesicles and begs the question as to whether the benefits of producing vesicles outweigh their costs. In this review, we discuss the various advantages and disadvantages associated with Gram-negative and Gram-positive bacterial vesicle production and offer perspective on the ultimate score. We also highlight questions needed to advance the field in determining the role for vesicles in bacterial survival, interkingdom communication, and virulence., (© 2021 The Authors.)

Leukotoxins are the critical virulence factors of several Gram-positive and Gram-negative bacteria [...].

Intravacuolar bacterial pathogens establish intracellular niches by constructing membrane-encompassed compartments. The vacuoles surrounding the bacteria are remarkably stable, facilitating microbial replication and preventing exposure to host cytoplasmically localized innate immune sensing mechanisms. To maintain integrity of the membrane compartment, the pathogen is armed with defensive weapons that prevent loss of vacuole integrity and potential exposure to host innate signaling. In some cases, the microbial components that maintain vacuolar integrity have been identified, but the basis for why the compartment degrades in their absence is unclear. In this review, we point out that lessons from the microbial-programmed degradation of the vacuole by the cytoplasmically localized Shigella flexneri provide crucial insights into how degradation of pathogen vacuoles occurs. We propose that in the absence of bacterial-encoded guard proteins, aberrant trafficking of host membrane-associated components results in a dysfunctional pathogen compartment. As a consequence, the vacuole is poisoned and replication is terminated., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Proteins have long been considered to be the most prominent factors regulating so-called invasive genes involved in host-pathogen interactions. The possible role of small non-coding RNAs (sRNAs), either intracellular, secreted or packaged in outer membrane vesicles (OMVs), remained unclear until recently. The advent of high-throughput RNA-sequencing (RNA-seq) techniques has accelerated sRNA discovery. RNA-seq radically changed the paradigm on bacterial virulence and pathogenicity to the point that sRNAs are emerging as an important, distinct class of virulence factors in both gram-positive and gram-negative bacteria. The potential of OMVs, as protectors and carriers of these functional, gene regulatory sRNAs between cells, has also provided an additional layer of complexity to the dynamic host-pathogen relationship. Using a non-exhaustive approach and through examples, this review aims to discuss the involvement of sRNAs, either free or loaded in OMVs, in the mechanisms of virulence and pathogenicity during bacterial infection. We provide a brief overview of sRNA origin and importance , and describe the classical and more recent methods of identification that have enabled their discovery, with an emphasis on the theoretical lower limit of RNA sizes considered for RNA sequencing and bioinformatics analyses.

Type III protein secretion systems (T3SSs), or injectisomes, are multiprotein nanomachines present in many Gram-negative bacteria that have a sustained long-standing close relationship with a eukaryotic host. These secretion systems have evolved to modulate host cellular functions through the activity of the effector proteins they deliver. To reach their destination, T3SS effectors must cross the multibarrier bacterial envelope and the eukaryotic cell membrane. Passage through the bacterial envelope is mediated by the needle complex, a central component of T3SSs that expands both the inner and outer membranes of Gram-negative bacteria. A set of T3SS secreted proteins, known as translocators, form a channel in the eukaryotic plasma membrane through which the effector proteins are delivered to reach the host cell cytosol. While the effector proteins are tailored to the specific lifestyle of the bacterium that encodes them, the injectisome is conserved among the different T3SSs. The central role of T3SSs in pathogenesis and their high degree of conservation make them a desirable target for the development of antimicrobial therapies against several important bacterial pathogens.

Intracellular bacterial pathogens intimately interact with the infected host cell to prevent elimination and to ensure survival. One group of intracellular pathogens, including Coxiella burnetii, Legionella pneumophila, Brucella spp., Anaplasma phagocytophilum, and Ehrlichia chaffeensis, utilizes a type IV secretion system (T4SS) that injects effectors to modulate host cell signalling, vesicular trafficking, autophagy, cell death and transcription to ensure survival [1]. So far, little emphasis has been directed towards understanding how these bacteria manipulate host cell metabolism. This manipulation is not only important for gaining access to nutrients, but also for regulating specific virulence programs [2,3]. Here, we will summarize recent progress made in characterizing the manipulation of host cell metabolism by C. burnetii and other intracellular pathogens utilizing a T4SS., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Signaling pathways allow bacteria to adapt to changing environments. For pathogenic bacteria, signaling pathways allow for timely expression of virulence factors and the repression of antivirulence factors within the mammalian host. As the bacteria exit the mammalian host, signaling pathways enable the expression of factors promoting survival in the environment and/or nonmammalian hosts. One such signaling pathway uses the dinucleotide cyclic-di-GMP (c-di-GMP), and many bacterial genomes encode numerous proteins that are responsible for synthesizing and degrading c-di-GMP. Once made, c-di-GMP binds to individual protein and RNA receptors to allosterically alter the macromolecule function to drive phenotypic changes. Each bacterial genome encodes unique sets of genes for c-di-GMP signaling and virulence factors so the regulation by c-di-GMP is organism specific. Recent works have pointed to evidence that c-di-GMP regulates virulence in different bacterial pathogens of mammalian hosts. In this review, we discuss the criteria for determining the contribution of signaling nucleotides to pathogenesis using a well-characterized signaling nucleotide, cyclic AMP (cAMP), in Pseudomonas aeruginosa. Using these criteria, we review the roles of c-di-GMP in mediating virulence and highlight common themes that exist among eight diverse pathogens that cause different diseases through different routes of infection and transmission. WIREs RNA 2018, 9:e1454. doi: 10.1002/wrna.1454 This article is categorized under: RNA in Disease and Development > RNA in Disease., (© 2017 Wiley Periodicals, Inc.)

In this review we examine the use of secretion systems by bacteria to subvert host functions. Bacteria have evolved multiple systems to interact with and overcome their eukaryotic host and other prokaryotes. Secretion systems are required for the release of several effectors through the bacterial membrane(s) into the extracellular space or directly into the cytoplasm of the host. We review the secretion systems of Gram-positive and Gram-negative bacteria and describe briefly the structural composition of the seven secretion systems that have been associated with increased virulence through subversion of host functions. Some of the effects of such systems on eukaryotic host processes have been studied extensively. We also describe the best-characterized effectors of each secretion system to give an overview of the molecular mechanisms employed by bacteria to hide from the immune system and convert eukaryotic cells into optimal ecological niches for their replication.

Noncoding RNAs (ncRNAs) regulating virulence have been identified in most pathogens. This review discusses RNA-mediated mechanisms exploited by bacterial pathogens to successfully infect and colonize their hosts. It discusses the most representative RNA-mediated regulatory mechanisms employed by two intracellular [Listeria monocytogenes and Salmonella enterica serovar Typhimurium (S. Typhimurium)] and two extracellular (Vibrio cholerae and Staphylococcus aureus) bacterial pathogens. We review the RNA-mediated regulators (e.g., thermosensors, riboswitches, cis- and trans-encoded RNAs) used for adaptation to the specific niches colonized by these bacteria (intestine, blood, or the intracellular environment, for example) in the framework of the specific pathophysiological aspects of the diseases caused by these microorganisms. A critical discussion of the newest findings in the field of bacterial ncRNAs shows how examples in model pathogens could pave the way for the discovery of new mechanisms in other medically important bacterial pathogens.

Gram-negative bacteria have mechanisms through which they can colonize and survive in different environments, such as the secretion systems types (1-6) that have been widely studied and characterized. Nowadays, some authors have proposed extracellular structures, such as the outer membrane vesicles (OMVs), to be considered as an additional and independent secretion system. The OMVs are spherical particles of 50-250 nm in diameter; they originate in the outer membrane, and therefore they have a very similar composition to the latter. These particles can transport an important variety of biomolecules: enzymes, toxins, antigenic determinants and even nucleic acids. Thus, it is of great interest to collect data describing the advantages of the transport of biomolecules through the OMVs and, thus, determine their role as a potential secretion system., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Type II secretion (T2S) is one means by which Gram-negative pathogens secrete proteins into the extracellular milieu and/or host organisms. Based upon recent genome sequencing, it is clear that T2S is largely restricted to the Proteobacteria , occurring in many, but not all, genera in the Alphaproteobacteria , Betaproteobacteria , Gammaproteobacteria , and Deltaproteobacteria classes. Prominent human and/or animal pathogens that express a T2S system(s) include Acinetobacter baumannii , Burkholderia pseudomallei , Chlamydia trachomatis , Escherichia coli , Klebsiella pneumoniae , Legionella pneumophila , Pseudomonas aeruginosa , Stenotrophomonas maltophilia , Vibrio cholerae , and Yersinia enterocolitica T2S-expressing plant pathogens include Dickeya dadantii , Erwinia amylovora , Pectobacterium carotovorum , Ralstonia solanacearum , Xanthomonas campestris , Xanthomonas oryzae , and Xylella fastidiosa T2S also occurs in nonpathogenic bacteria, facilitating symbioses, among other things. The output of a T2S system can range from only one to dozens of secreted proteins, encompassing a diverse array of toxins, degradative enzymes, and other effectors, including novel proteins. Pathogenic processes mediated by T2S include the death of host cells, degradation of tissue, suppression of innate immunity, adherence to host surfaces, biofilm formation, invasion into and growth within host cells, nutrient assimilation, and alterations in host ion flux. The reach of T2S is perhaps best illustrated by those bacteria that clearly use it for both environmental survival and virulence; e.g., L. pneumophila employs T2S for infection of amoebae, growth within lung cells, dampening of cytokines, and tissue destruction. This minireview provides an update on the types of bacteria that have T2S, the kinds of proteins that are secreted via T2S, and how T2S substrates promote infection., (Copyright © 2017 American Society for Microbiology.)

Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted "effector" proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Outer membrane vesicles (OMVs) are spherical nanostructures that are ubiquitously shed from gram-negative bacteria both in vitro and in vivo. Recent findings revealed that OMVs, which contain diverse components derived from the parent bacterium, play an important role in communication with neighboring bacteria and the environment. Furthermore, nanoscale proteoliposomes decorated with pathogen-associated molecules attract considerable attention as a non-replicative carrier for vaccines and drug materials. This review introduces recent advances in OMV biogenesis and discusses the roles of OMVs in the context of bacterial communication and virulence regulation. It also describes the remarkable accomplishments in OMV engineering for diverse therapeutic applications.

Biochemical studies suggest that the NAIP family of NLR proteins are cytosolic innate receptors that directly recognize bacterial ligands and trigger NLRC4 inflammasome activation. In this study, we generated Naip5(-/-), Naip1(-/-), and Naip2(-/-) mice and showed that bone marrow macrophages derived from these knockout mice are specifically deficient in detecting bacterial flagellin, the type III secretion system needle, and the rod protein, respectively. Naip1(-/-), Naip2(-/-), and Naip5(-/-) mice also resist lethal inflammasome activation by the corresponding ligand. Furthermore, infections performed in the Naip-deficient macrophages have helped to define the major signal in Legionella pneumophila, Salmonella Typhimurium and Shigella flexneri that is detected by the NAIP/NLRC4 inflammasome. Using an engineered S. Typhimurium infection model, we demonstrate the critical role of NAIPs in clearing bacterial infection and protecting mice from bacterial virulence-induced lethality. These results provide definitive genetic evidence for the important physiological function of NAIPs in antibacterial defense and inflammatory damage-induced lethality in mice., (© 2016 Zhao et al.)

The actin cytoskeleton is a key target of numerous microbial pathogens, including protozoa, fungi, bacteria and viruses. In particular, bacterial pathogens produce and deliver virulence effector proteins that hijack actin dynamics to enable bacterial invasion of host cells, allow movement within the host cytosol, facilitate intercellular spread or block phagocytosis. Many of these effector proteins directly or indirectly target the major eukaryotic actin nucleator, the Arp2/3 complex, by either mimicking nucleation promoting factors or activating upstream small GTPases. In contrast, this review is focused on a recently identified class of effector proteins from Gram-negative bacteria that function as direct actin nucleators. These effector proteins mimic functional activities of formins, WH2-nucleators and Ena/VASP assembly promoting factors demonstrating that bacteria have coopted the complete set of eukaryotic actin assembly pathways. Structural and functional analyses of these nucleators have revealed several motifs and/or mechanistic activities that are shared with eukaryotic actin nucleators. However, functional effects of these proteins during infection extend beyond plain actin polymerization leading to interference with other host cell functions such as vesicle trafficking, cell cycle progression and cell death. Therefore, their use as model systems could not only help in the understanding of the mechanistic details of actin polymerization but also provide novel insights into the connection between actin dynamics and other cellular pathways., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Many Gram-negative bacterial pathogens use the type III secretion system to deliver effector proteins into host cells. These effectors use various mechanisms to exploit host processes to the advantage of the pathogen. A large group of effectors use post-translational modifications, either reversible or irreversible, to manipulate host proteins, and while most of these mechanisms mimic eukaryotic activities, others appear to be unique biochemical functions. Deciphering such mechanisms and identifying the host targets of these effectors sheds light on eukaryotic signaling pathways and immune responses., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The innate immune system provides the first line of host defence against invading pathogens. Key to upregulation of the innate immune response are Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns (PAMPs) and trigger a signaling pathway culminating in the production of inflammatory mediators. Central to this TLR signaling pathway are heterotypic protein-protein interactions mediated through Toll/interleukin-1 receptor (TIR) domains found in both the cytoplasmic regions of TLRs and adaptor proteins. Pathogenic bacteria have developed a range of ingenuous strategies to evade the host immune mechanisms. Recent work has identified a potentially novel evasion mechanism involving bacterial TIR domain proteins. Such domains have been identified in a wide range of pathogenic bacteria, and there is evidence to suggest that they interfere directly with the TLR signaling pathway and thus inhibit the activation of NF-κB. The individual TIR domains from the pathogenic bacteria Salmonella enterica serovar Enteritidis, Brucella sp, uropathogenic E. coli and Yersinia pestis have been analyzed in detail. The individual bacterial TIR domains from these pathogenic bacteria seem to differ in their modes of action and their roles in virulence. Here, we review the current state of knowledge on the possible roles and mechanisms of action of the bacterial TIR domains.

Bacterial protein toxins are important virulence factors. A particular class of toxins, the pore-form toxins (PFTs), shares the toxigenic mechanism of forming pores in the membrane of target cells. The relationship between autophagy and bacterial PFTs has been described for several toxin-secreting pathogens and in this review we have recapitulated the more recent findings on this issue. A common outcome is that the target cell, by a yet non-completely defined mechanism, senses the toxin attack and builds up complex responses as a protective mechanism for host survival. However, in some cases, this cellular response is beneficial to the microorganism by supplying an intracellular niche or by promoting host-cell death, which facilitates pathogen spreading.

Protein toxins are important virulence factors contributing to neonatal sepsis. The major pathogens of neonatal sepsis, group B Streptococci, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus, secrete toxins of different molecular nature, which are key for defining the disease. Amongst these toxins are pore-forming exotoxins that are expressed as soluble monomers prior to engagement of the target cell membrane with subsequent formation of an aqueous membrane pore. Membrane pore formation is not only a means for immediate lysis of the targeted cell but also a general mechanism that contributes to penetration of epithelial barriers and evasion of the immune system, thus creating survival niches for the pathogens. Pore-forming toxins, however, can also contribute to the induction of inflammation and hence to the manifestation of sepsis. Clearly, pore-forming toxins are not the sole factors that drive sepsis progression, but they often act in concert with other bacterial effectors, especially in the initial stages of neonatal sepsis manifestation.

The recent and dramatic rise of antibiotic resistance among bacterial pathogens underlies the fear that standard treatments for infectious disease will soon be largely ineffective. Resistance has evolved against nearly every clinically used antibiotic, and in the near future, we may be hard-pressed to treat bacterial infections previously conquered by "magic bullet" drugs. While traditional antibiotics kill or slow bacterial growth, an important emerging strategy to combat pathogens seeks to block the ability of bacteria to harm the host by inhibiting bacterial virulence factors. One such virulence factor, the type three secretion system (T3SS), is found in over two dozen Gram-negative pathogens and functions by injecting effector proteins directly into the cytosol of host cells. Without T3SSs, many pathogenic bacteria are unable to cause disease, making the T3SS an attractive target for novel antimicrobial drugs. Interdisciplinary efforts between chemists and microbiologists have yielded several T3SS inhibitors, including the relatively well-studied salicylidene acylhydrazides. This review highlights the discovery and characterization of T3SS inhibitors in the primary literature over the past 10 years and discusses the future of these drugs as both research tools and a new class of therapeutic agents.

A paradigm shift in our thinking about the intricacies of the host-parasite interaction is required that considers bacterial structures and their relationship to bacterial pathogenesis. It has been proposed that interactions between extended macromolecular assemblies, termed hyperstructures (which include multiprotein complexes), determine bacterial phenotypes. In particular, it has been proposed that hyperstructures can alter virulence. Two such hyperstructures have been characterized in both pathogenic and nonpathogenic bacteria. Present within a number of both human and plant Gram-negative pathogens is the type 3 secretion system (T3SS) injectisome which in some bacteria serves to inject toxic effector proteins directly into targeted host cells resulting in their paralysis and eventual death (but which in other bacteria prevents the death of the host). The injectisome itself comprises multiple protein subunits, which are all essential for its function. The degradosome is another multiprotein complex thought to be involved in cooperative RNA decay and processing of mRNA transcripts and has been very well characterized in nonpathogenic Escherichia coli. Recently, experimental evidence has suggested that a degradosome exists in the yersiniae as well and that its interactions within the pathogens modulate their virulence. Here, we explore the possibility that certain interactions between hyperstructures, like the T3SS and the degradosome, can ultimately influence the virulence potential of the pathogen based upon the physical locations of hyperstructures within the cell.

Many bacterial pathogens employ multicomponent protein complexes to deliver macromolecules directly into their eukaryotic host cell to promote infection. Some Gram-negative pathogens use a versatile Type IV secretion system (T4SS) that can translocate DNA or proteins into host cells. T4SSs represent major bacterial virulence determinants and have recently been the focus of intense research efforts designed to better understand and combat infectious diseases. Interestingly, although the two major classes of T4SSs function in a similar manner to secrete proteins, the translocated 'effectors' vary substantially from one organism to another. In fact, differing effector repertoires likely contribute to organism-specific host cell interactions and disease outcomes. In this review, we discuss the current state of T4SS research, with an emphasis on intracellular bacterial pathogens of humans and the diverse array of translocated effectors used to manipulate host cells.

Whether they live in the soil, drift in the ocean, survive in the lungs of human hosts or reside on the surfaces of leaves, all bacteria must cope with an array of environmental stressors. Bacteria have evolved an impressive suite of protein secretion systems that enable their survival in hostile environments and facilitate colonization of eukaryotic hosts. Collectively, gram-negative bacteria produce six distinct secretion systems that deliver proteins to the extracellular milieu or directly into the cytosol of host cells. The type VI secretion system (T6SS) was discovered recently and is encoded in at least one fourth of all sequenced gram-negative bacterial genomes. T6SS proteins are evolutionarily and structurally related to phage proteins, and it is likely that the T6SS apparatus is reminiscent of phage injection machinery. Most studies of T6SS function have been conducted in the context of host-pathogen interactions. However, the totality of data suggests that the T6SS is a versatile tool with roles in virulence, symbiosis, interbacterial interactions, and antipathogenesis. This review gives a brief history of T6SS discovery and an overview of the pathway's predicted structure and function. Special attention is paid to research addressing the T6SS of plant-associated bacteria, including pathogens, symbionts and plant growth-promoting rhizobacteria.

Gram-negative bacterial pathogens of humans have evolved a range of virulence factors to promote motility, attach to epithelial or endothelial cell surfaces, avoid host immune responses, activate or inactivate host cellular pathways and ultimately cause clinical disease. Gram-negative sepsis is a life-threatening complication of these events. This review discusses the virulence factors of common Gram-negative bacteria causing human sepsis with a focus on Neisseria meningitidis. Adherence, motility, colonization and cell entry involve bacterial pili, flagella and outer membrane proteins. Endotoxin (lipopoly-or lipo-oligosaccharide), other membrane components or exotoxins can be potent inducers of the host inflammatory cascade via innate receptor pathways. Capsular polysaccharides and outer membrane proteins can help the bacterium evade immune defenses. The role in pathogenesis of iron acquisition, bacterial secretion systems, quorum sensing, and biofilm formation is also reviewed. Through multiple genetic mechanisms leading to phase variation, Gram-negative bacteria can adapt to changing host and environmental conditions and selective pressures. Further, the antimicrobial resistance of Gram-negative bacteria driven by antibiotic use will continue to influence the clinical outcomes of Gram-negative sepsis in the coming years., (Copyright © 2011 S. Karger AG, Basel.)

Outer membrane vesicles (OMVs), ubiquitously shed from Gram-negative bacteria, contain various virulence factors such as toxins, proteases, adhesins, and lipopolysaccharide, which are utilized to establish a colonization niche, modulate host defense and response, and impair host cell function. Thus, OMVs can be considered as a type of bacterial offensive weapon. This review discusses the entry mechanism of OMVs into host cells as well as their etiological roles in host-parasite interactions., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

Type IV secretion systems (T4SSs) are large protein complexes which traverse the cell envelope of many bacteria. They contain a channel through which proteins or protein-DNA complexes can be translocated. This translocation is driven by a number of cytoplasmic ATPases which might energize large conformational changes in the translocation complex. The family of T4SSs is very versatile, shown by the great variety of functions among family members. Some T4SSs are used by pathogenic Gram-negative bacteria to translocate a wide variety of virulence factors into the host cell. Other T4SSs are utilized to mediate horizontal gene transfer, an event that greatly facilitates the adaptation to environmental changes and is the basis for the spread of antibiotic resistance among bacteria. Here we review the recent advances in the characterization of the architecture and mechanism of substrate transfer in a few representative T4SSs with a particular focus on their diversity of structure and function.

The aim of the current study was to inhibit quoring-sensing(QS)-mediated virulence factors of representative Gram-negative bacteria by marine bacterial isolates. Bacteria isolated from Palk Bay sediments were screened for anti-QS activity. Eleven strains inhibited QS signals in Chromobacterium violaceum (ATCC 12472) and C. violaceum CV026. The marine bacterial strain S8-07 reduced the accumulation of N-acyl homoserine lactone (AHLs) and showed significant inhibition of LasA protease(76%), LasB elastase(84%), caseinase(70%), pyocyanin (84%), pyoverdin and biofilm formation(87%) in Pseudomonas aeruginosa PAO1. Strain S8-07 also showed highly significant reduction (90%) in prodigiosin, secreted casienase (92%), hemolytic activity (73%) and biofilm formation (61%) in Serratia marcescens. Strain S8-07, identified as Bacillus pumilus (accession number FJ584416), showed distinct profiles of inhibition against the virulence factors of both P. aeruginosa PAO1 (las, rhl) and S. marcescens (shl). Polar extraction and proteinase K treatment of the culture supernatant confirmed that the anti-QS activity of S8-07 was indeed due to a protein molecule. Acidification assay and HPLC analysis revealed that the degradation of AHL was not due to lactonase activity, but rather, was due to acylase activity of S8-07. Thus, novel anti-QS acylase activity is reported for the first time from a B. pumilus strain of marine origin., ((c) 2010 Elsevier Masson SAS. All rights reserved.)

Microorganisms have developed several mechanisms to modulate the host immune system to increase their survival and propagation in the host. Their presence in the host is not only revealed by self-produced peptides but also through host-derived chemokines and active complement fragments. These so-called chemoattractants are recognized by G protein-coupled receptors (GPCRs) expressed on leukocyte cell membranes. Activation of GPCRs triggers leukocyte activation and guides their recruitment to the site of infection. Therefore, GPCRs play a central role in leukocyte trafficking leading to microbial clearance. It is therefore not surprising that microorganisms are able to sabotage this arm of the immune response. Different microorganisms have evolved a variety of tactics to modulate GPCR activation. Here, we review the mechanisms and proteins used by major human pathogens and less virulent microorganisms that affect GPCR signaling. While viruses generally produce receptor and chemoattractant mimics, parasites and bacteria such as Staphylococcus aureus, Streptococcus pyogenes, Porphyromonas gingivalis, and Bordetella pertussis secrete proteins that affect receptor signaling, directly antagonize receptors, cleave stimuli, and even prevent stimulus generation. As the large arsenal of GPCR modulators aids prolonged microbial persistence in the host, their study provides us a better understanding of microbial pathogenesis., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The Type III secretion system (TTSS) facilitates the export of effector proteins from pathogenic and symbiotic Gram-negative bacteria into the cytosol of eukaryotic host cells. The current functional and evolutionary knowledge on the molecular recognition of TTSS substrates and computational models of the secretion signal are discussed in this review., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

Rapid diagnosis and treatment of disease is often based on the identification and characterization of causative agents derived from phenotypic characteristics. Current methods can be laborious and time-consuming, often requiring many skilled personnel and a large amount of lab space. The objective of our study was to develop a spotted microarray for rapid identification and characterization of bacterial pathogens and their antimicrobial resistance genes. Our spotted microarray consists of 489 70mer probes that detect 40 bacterial pathogens of medical, veterinary and zoonotic importance (including 15 NIAID Category A, B and C pathogens); associated genes that encode resistance for antimicrobial and metal resistance; and DNA elements that are important for horizontal gene transfer among bacteria. High specificity and reliability of the microarray was achieved for bacterial pathogens of animal and human importance by validating MDR pathogenic bacteria as pure cultures or by following their inoculation in complex and highly organic sample matrices, such as soil and manure., (Copyright 2010 Elsevier B.V. All rights reserved.)

Proteomic technology offers an unprecedented systematic approach to investigate the protein complement of any organism. The field of microbial pathogenesis has greatly benefited from other systems approaches, and the application of proteomics to the study of infectious agents is beginning to emerge. Such applications include unambiguously identifying complete virulence factor inventories, studying the response of both host and pathogen to the infection process and elucidating mechanistic actions of virulence factors as they interface with host cells. This review will highlight examples where proteomic studies have contributed to our understanding of pathogenesis in these areas, with an emphasis on pathogens that employ type III and type IV secretion systems. In addition, we will discuss areas where proteomics may help shape further investigation and discovery in this field.

Type IV secretion systems (T4SSs) are important virulence factors used by Gram-negative bacterial pathogens to inject effectors into host cells or to spread plasmids harboring antibiotic resistance genes. We report the 15 angstrom resolution cryo-electron microscopy structure of the core complex of a T4SS. The core complex is composed of three proteins, each present in 14 copies and forming a approximately 1.1-megadalton two-chambered, double membrane-spanning channel. The structure is double-walled, with each component apparently spanning a large part of the channel. The complex is open on the cytoplasmic side and constricted on the extracellular side. Overall, the T4SS core complex structure is different in both architecture and composition from the other known double membrane-spanning secretion system that has been structurally characterized.

Bacterial virulence mechanisms are attractive targets for antibiotic development because they are required for the pathogenesis of numerous global infectious disease agents. The bacterial secretion systems used to assemble the surface structures that promote adherence and deliver protein virulence effectors to host cells could comprise one such therapeutic target. In this study, we developed and performed a high-throughput screen of small molecule libraries and identified one compound, a 2-imino-5-arylidene thiazolidinone that blocked secretion and virulence functions of a wide array of animal and plant Gram-negative bacterial pathogens. This compound inhibited type III secretion-dependent functions, with the exception of flagellar motility, and type II secretion-dependent functions, suggesting that its target could be an outer membrane component conserved between these two secretion systems. This work provides a proof of concept that compounds with a broad spectrum of activity against Gram-negative bacterial secretion systems could be developed to prevent and treat bacterial diseases.

The extracellular products (ECP) secreted by two strains of gram-negative bacteria isolated from Nephrops norvegicus exhibiting signs of an opportunistic bacterial infection were investigated with the objective of understanding their role in the spoilage of host muscle tissue and identifying disease related virulence mechanisms. ECP from Vibrio sp. demonstrated no proteolytic activity. ECP from Pseudoalteromonas sp. (isolate N10) degraded several substrates, including azocasein and host muscle tissue. Proteolytic activity increased with temperature. Substrate-impregnated sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the effect of the isolates' ECP on the molecular weight of proteins derived from abdominal muscle tissue revealed that the ECP of Pseudoalteromonas sp. selectively degraded the myosin heavy chain, troponin-T, troponin-I, paramyosin and several unidentified muscle proteins approximately 110 kDa in size. Topomyosin was also reduced in quantity. Degradation of SDS-PAGE gels impregnated with host muscle proteins, by the ECP of Pseudoalteromonas sp. revealed 3 zones of proteolysis, with estimated molecular weights between 100 and 30 kDa, indicating multiple proteases in the ECP. Through the API ZYM system, both isolates demonstrated strong leucine arylamidase activity, with the Vibrio sp. showing strong acid phosphatase activity. These enzymes have been identified as disease related virulence mechanisms in other bacterial pathogens. There is likely a complex pathway to the final condition, involving virulence factors of other species and the stresses involved in capture and transport.

Multiple pathogenic Gram-negative bacteria produce cytolethal-distending toxins (CDTs). CDT is typically composed of three subunits: the catalytic subunit CdtB has DNase I-like activity, whereas CdtA and CdtC are binding proteins for delivering CdtB into target cells. Translocation of CdtB to the nucleus induces genotoxic effects on host DNA, triggering DNA repair cascades that lead to cell cycle arrest and eventual cell death. Several lines of evidence indicate that this toxin contributes to the pathogenicity of CDT-producing bacteria in vivo. Helicobacter hepaticus and Campylobacter jejuni CDTs are essential for persistent infection of the gastrointestinal tract and increase the severity of mucosal inflammation or liver disease in susceptible mouse strains. Haemophilus ducreyi CDT may contribute to the pathogenesis of chancroid in rabbits. Recently, H. hepaticus CDT has been shown to play a crucial role in promoting the progression of infectious hepatitis to pre-malignant, dysplastic lesions via activation of a pro-inflammatory NF-kappaB pathway and increased proliferation of hepatocytes, providing the first evidence that CDT has carcinogenic potential in vivo. Thus, both in vitro and in vivo data indicate that CDT is a bacterial virulence factor.

In recent years mounting problems related to antibiotic-resistant bacteria have resulted in the prediction that we are entering the preantibiotic era. A way of preventing such a development would be to introduce novel antibacterial medicines with modes of action distinct from conventional antibiotics. Recent studies of bacterial virulence factors and toxins have resulted in increased understanding of the way in which pathogenic bacteria manipulate host cellular processes. This knowledge may now be used to develop novel antibacterial medicines that disarm pathogenic bacteria. The type III secretion system (T3SS) is known to be a potent virulence mechanism shared by a broad spectrum of pathogenic Gram-negative bacteria that interact with human, animal and plant hosts by injecting effector proteins into the cytosol of host cells. Diseases, such as bubonic plague, shigellosis, salmonellosis, typhoid fever, pulmonary infections, sexually transmitted chlamydia and diarrhoea largely depend on the bacterial proteins injected by the T3SS machinery. Recently a number of T3SS inhibitors have been identified using screening-based approaches. One class of inhibitors, the salicylidene acylhydrazides, has been subjected to chemical optimization and evaluation in several in vitro and ex vivo assays in multiple bacterial species including Yersinia spp., Chlamydia spp., Salmonella spp. and Pseudotuberculosis aeruginosa. Reports published up to date indicate that T3SS inhibitors have the potential to be developed into novel antibacterial therapeutics.

N(6) methylation in adenosine moieties causes changes in DNA structure and can modulate DNA-protein interactions. In both alpha-Proteobacteria and gamma-Proteobacteria, postreplicative formation of N(6)-methyl-adenine regulates transcription of specific genes and provides two general types of controls: (i) clock-like controls that permit transient gene transcription during a specific stage of DNA replication; (ii) switch-like controls in which transcription is regulated by a DNA methylation pattern. DNA adenine methylation may also regulate gene expression by affecting nucleoid topology. Recent transcriptomic studies have unveiled novel cases of genes regulated by DNA adenine methylation, including virulence genes of bacterial pathogens.

Pathogenic bacteria employ many strategies to overcome the host immune system for extended survival and propagation in their hosts. Components of the bacterial outer-membrane play an important role in this process. When invading the host, Gram-negative bacteria often use a strategy, known as phase variation, that involves a reversible change in antigenic determinants, frequently polysaccharides. This means that the genes encoding the outer-membrane antigens undergo reversible changes within repeated simple DNA sequence motifs. The antigenic structure of the bacterial outer-membrane is influenced by the character of the host immune system, as well as by the targets for bacterial invasion. When the selection pressure of the immune system is absent or weak, bacteria can fail to synthesise the outer-membrane antigens, which are not needed at that time. Smooth-to-rough (S-R) mutation, an economical and often irreversible process in some Gram-negative bacteria, involves the gradual shortening of the lipopolysaccharide (LPS) O-chain. Under certain conditions, e.g., propagation in embryonated eggs or cell lines, some bacteria will cease synthesis of the complete LPS O-chain because it is an energy-demanding process. A type of gradual shortening of the LPS O-chain by Coxiella burnetii, traditionally called phase variation, is used in serological tests for the diagnosis of Q fever. This review discusses the role and function of polysaccharides, especially LPS produced by some Gram-negative bacteria, in bacterial survival.

Type VI secretion is a newly described mechanism for protein transport across the cell envelope of Gram-negative bacteria. Components that have been partially characterised include an IcmF homologue, the ATPase ClpV, a regulatory FHA domain protein and the secreted VgrG and Hcp proteins. Type VI secretion is clearly a key virulence factor for some important pathogenic bacteria and has been implicated in the translocation of a potential effector protein into eukaryotic cells by at least one organism (Vibrio cholerae). However, type VI secretion systems (T6SSs) are widespread in nature and not confined to known pathogens. In accordance with the general rule that the expression of protein secretion systems is tightly regulated, expression of type VI secretion is controlled at both transcriptional and post-transcriptional levels.

Bacterial virulence factors enable a host to replicate and disseminate within a host in part by subverting or eluding host defenses. The use of genomic techniques has led to the identification of new virulence factors that may serve as targets for new therapies. These putative virulence factors must be rigorously evaluated with in vitro and in vivo studies with an awareness of the technical limitations of each approach as well as an assessment of the prevalence of this factor in clinical bacterial isolates retrieved from appropriately controlled epidemiologic studies.

Type III secretion systems (T3SSs) are complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism enabling them to inject bacterial effector proteins directly into the host cell cytoplasm, bypassing the extracellular milieu. Although the effector proteins vary among different T3SS pathogens, common pathogenic mechanisms emerge, including interference with the host cell cytoskeleton to promote attachment and invasion, interference with cellular trafficking processes, cytotoxicity and barrier dysfunction, and immune system subversion. The activity of the T3SSs correlates closely with infection progression and outcome, both in animal models and in human infection. Therefore, to facilitate patient care and improve outcomes, it is important to understand the T3SS-mediated virulence processes and to target T3SSs in therapeutic and prophylactic development efforts.

Diverse pathogenic bacteria have developed similar mechanisms to subvert host cell responses. In this Progress article, we focus on bacterial virulence factors with different enzymatic activities that can increase the expression of the Kruppel-like factor (KLF) family of mammalian transcriptional regulators through their ability to modify the activity of a common host-cell target - the Rho protein family. By using a common virulence strategy, both Gram-negative and Gram-positive pathogens exploit the KLF regulatory cascade to modulate nuclear factor kappaB activation, pro-inflammatory cytokine expression, actin cytoskeletal dynamics and phagocytosis.

Type IV secretion systems are used by many gram-negative bacteria for the translocation of macromolecules (proteins, DNA, or DNA-protein complexes) across the cell envelope. Among them are many pathogens for which type IV secretion systems are essential virulence factors. Type IV secretion systems comprise 8-12 conserved proteins, which assemble into a complex spanning the inner and the outer membrane, and many assemble extracellular appendages, such as pili, which initiate contact with host and recipient cells followed by substrate translocation. VirB8 is an essential assembly factor for all type IV secretion systems. Biochemical, cell biological, genetic, and yeast two-hybrid analyses showed that VirB8 undergoes multiple interactions with other type IV secretion system components and that it directs polar assembly of the membrane-spanning complex in the model organism Agrobacterium tumefaciens. The availability of the VirB8 X-ray structure has enabled a detailed structure-function analysis, which identified sites for the binding of VirB4 and VirB10 and for self-interaction. Due to its multiple interactions, VirB8 is an excellent model for the analysis of assembly factors of multiprotein complexes. In addition, VirB8 is a possible target for drugs that target its protein-protein interactions, which would disarm bacteria by depriving them of their essential virulence functions.

Many bacterial pathogens employ a type III secretion system to deliver type III secreted effectors (T3SEs) into host cells, where they interact directly with host substrates to modulate defense pathways and promote disease. This interaction creates intense selective pressures on these secreted effectors, necessitating rapid evolution to overcome host surveillance systems and defenses. Using computational and evolutionary approaches, we have identified numerous mosaic and truncated T3SEs among animal and plant pathogens. We propose that these secreted virulence genes have evolved through a shuffling process we have called "terminal reassortment." In terminal reassortment, existing T3SE termini are mobilized within the genome, creating random genetic fusions that result in chimeric genes. Up to 32% of T3SE families in species with relatively large and well-characterized T3SE repertoires show evidence of terminal reassortment, as compared to only 7% of non-T3SE families. Terminal reassortment may permit the near instantaneous evolution of new T3SEs and appears responsible for major modifications to effector activity and function. Because this process plays a more significant role in the evolution of T3SEs than non-effectors, it provides insight into the evolutionary origins of T3SEs and may also help explain the rapid emergence of new infectious agents.

Cytolethal distending toxin (CDT) is a bacterial toxin that initiates a eukaryotic cell cycle block at the G2 stage prior to mitosis. CDT is produced by a number of bacterial pathogens including: Campylobacter species, Escherichia coli, Salmonella enterica serovar Typhi, Shigella dystenteriae, enterohepatic Helicobacter species, Actinobacillus actinomycetemcomitans (the cause of aggressive periodontitis), and Haemophilus ducreyi (the cause of chancroid). The functional toxin is composed of three proteins; CdtB potentiates a cascade leading to cell cycle block, and CdtA and CdtC function as dimeric subunits, which bind CdtB and delivers it to the mammalian cell interior. Once inside the cell, CdtB enters the nucleus and exhibits a DNase I-like activity that results in DNA double-strand breaks. The eukaryotic cell responds to the DNA double-strand breaks by initiating a regulatory cascade that results in cell cycle arrest, cellular distension, and cell death. Mutations in CdtABC that cause any of the three subunits to lose function prevent the bacterial cell from inducing cytotoxicity. The result of CDT activity can differ somewhat depending on the eukaryotic cell types affected. Epithelial cells, endothelial cells, and keratinocytes undergo G2 cell cycle arrest, cellular distension, and death; fibroblasts undergo G1 and G2 arrest, cellular distension, and death; and immune cells undergo G2 arrest followed by apoptosis. CDT contributes to pathogenesis by inhibiting both cellular and humoral immunity via apoptosis of immune response cells, and by generating necrosis of epithelial-type cells and fibroblasts involved in the repair of lesions produced by pathogens resulting in slow healing and production of disease symptoms. Thus, CDT may function as a virulence factor in pathogens that produce the toxin.

Phospholipase A (PLA) is one of the few enzymes present in the outer membrane of Gram-negative bacteria, and is likely to be involved in the membrane disruption processes that occur during host cell invasion. Both secreted and membrane-bound phospholipase A(2) activities have been described in bacteria, fungi and protozoa. Recently there have been increasing reports on the involvement of PLA in bacterial invasion and pathogenesis. This review highlights the latest findings on PLA as a virulence factor in Gram-negative bacteria.

Bacterial type III secretion systems are thought to translocate virulence proteins directly from the bacterial cytoplasm into host cells through a continuous molecular channel. Little is known about how the apparatus itself interacts with membranes and whether insertion of this structure into the host membrane has consequences for the bacteria apart from its beneficial role in delivering virulence proteins. New evidence suggests that membrane insertion of the bacterial type III apparatus might turn on a calcium-dependent signaling pathway resulting in phagolysosomal fusion.