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Two unanswered questions in the field of Chlamydia are 1) How does the inclusion grow over time? and 2) What promotes RB-to-EB conversion? Small molecule compounds have been widely used to manipulate infection conditions to determine specific targets required for these stages of chlamydial development. Although there are many publications describing the effects of anti-chlamydial inhibitors, the lack of a standard approach makes it challenging to compare these effects across studies.In chapter 3, our manuscript (submitted) defines the weaknesses in the current methodologies that the Chlamydia field implements to study inhibitor treatments during the infection. We additionally demonstrate approaches to yield more information from inhibitor studies to more accurately describe the ways different conditions alter the infection. Using published and novel chlamydial inhibitors, we found that analysis with one assay at one or two time points in the infection is insufficient because inhibitors can change the length of the infection. Furthermore, we showed that phenotypes can vary depending on the time of analysis and the assay used. To circumvent these issues, we propose a novel measurement, Progenymax, which takes changes in the length of the developmental cycle into consideration and can easily be used to compare differences in progeny, regardless of the condition.In chapter 4, we describe a novel inhibitor of the chlamydial infection, H89, which slowed inclusion growth and caused a block in RB-to-EB conversion. We tested several known kinases and pathways that were previously reported to be blocked by H89, but none were inhibited by our H89 treatment conditions. In chapter 5, we continued to explore the mechanisms targeted by H89 to elucidate why the inhibitor was such a strong anti-chlamydial inhibitor. We found that H89 causes lysosomal accumulation of cholesterol, which correlated with a defect in lysosomal acidification. These studies suggest that H89 prevents lysosomal acidification, resulting in cholesterol accumulation. Previous studies suggest that cholesterol may be necessary for the chlamydial infection, meaning these H89-mediated defects on inclusion growth and progeny production are likely due to a decrease in cholesterol transport out of the lysosome and reduced cholesterol accessibility by chlamydiae.

Enterococcus faecalis (E. faecalis) biofilms are implicated in endocarditis, urinary tract infections, and biliary tract infections. Coupled with E. faecalis internalization into host cells, this opportunistic pathogen poses great challenges to conventional antibiotic therapy. The inability of ampicillin (Amp) to eradicate bacteria hidden in biofilms and intracellular niches greatly reduces its efficacy against complicated E. faecalis infections. To enhance the potency of Amp against different forms of E. faecalis infections, Amp was loaded into Lipid-Polymer hybrid Nanoparticles (LPNs), a highly efficient nano delivery platform consisting of a unique combination of DOTAP lipid shell and PLGA polymeric core. The antibacterial activity of these nanoparticles (Amp-LPNs) was investigated in a protozoa infection model, achieving a much higher multiplicity of infection (MOI) compared with studies using animal phagocytes. A significant reduction of total E. faecalis was observed in all groups receiving 250 µg/mL Amp-LPNs compared with groups receiving the same concentration of free Amp during three different interventions, simulating acute and chronic infections and prophylaxis. In early intervention, no viable E. faecalis was observed after 3 h LPNs treatment whereas free Amp did not clear E. faecalis after 24 h treatment. Amp-LPNs also greatly enhanced the antibacterial activity of Amp at late intervention, and boosted the survival rate of protozoa approaching 400%, where no viable protozoa were identified in the free Amp groups at the 40 h post-infection treatment timepoint. Prophylactic effectiveness with Amp-LPNs at a concentration of 250 μg/mL was exhibited in both bacteria elimination and protozoa survival towards subsequent infections. Using protozoa as a surrogate model for animal phagocytes to study high MOI infections, this study suggests that LPN-formulated antibiotics hold the potential to significantly improve the therapeutic outcome in highly complicated bacterial infections. Agri-Food and Veterinary Authority of Singapore (AVA) Ministry of Education (MOE) Accepted version The authors would like to acknowledge the financial support from the Singapore Centre for Environmental Life Sciences Engineering (SCELSE) (MOE/RCE: M4330019.C70), Ministry of Education AcRF-Tier 1 grant (RG19/18), Agri-Food & Veterinary Authority of Singapore (APF LCK102), Biomedical Research Council (BMRC) - Therapeutics Development Review (TDR-G-004-001), NTU-HSPH grant (NTU-HSPH 17002), and the Bill and Melinda Gates Foundation (OPP1199116).

Cell surface nucleotide-metabolizing enzyme, ectonucleotidase-CD73, has emerged as a central component of the cellular homeostatic-machinery that counterbalances the danger-molecule (extracellular-ATP)-driven proinflammatory response in immune cells. While the importance of CD73 in microbial host fitness and symbiosis is gradually being unraveled, there remains a significant gap in knowledge of CD73 and its putative role in epithelial cells. Here, we depict a novel host-pathogen adaptation mechanism where CD73 takes a center role in the intracellular persistence of Porphyromonas gingivalis, a major colonizer of oral mucosa, using human primary gingival epithelial cell (GEC) system. Temporal analyses revealed, upon invasion into the GECs, P. gingivalis can significantly elevate the host-surface CD73 activity and expression. The enhanced and active CD73 significantly increases P. gingivalis intracellular growth in the presence of substrate-AMP and simultaneously acts as a negative regulator of reactive oxygen species (ROS) generation upon eATP treatment. The inhibition of CD73 by siRNA or by a specific inhibitor markedly increases ROS production. Moreover, CD73 and P. gingivalis cross-signaling significantly modulates pro-inflammatory interleukin-6 (IL-6) in the GECs. Conversely, exogenous treatment of the infected GECs with IL-6 suppresses the intracellular bacteria via amplified ROS generation. However, the decreased bacterial levels can be restored by overexpressing functionally active CD73. Together, these findings illuminate how the local extracellular-purine-metabolism, in which CD73 serves as a core molecular switch, can alter intracellular microbial colonization resistance. Further, host-adaptive pathogens such as P. gingivalis can target host ectonucleotidases to disarm specific innate defenses for successful intracellular persistence in mucosal epithelia.

[Aim]: First, to compare in vitro minimum inhibitory concentrations (MIC) of free cloxacillin and cloxacillin-containing nanoparticles (NP) against methicillin-susceptible (MSSA) and resistant Staphylococcus aureus (MRSA) and second, to assess NP antimicrobial activity against intracellular S. aureus., [Methods]: Poly(d,l-lactide-co-glycolide) acid (PLGA)-NP were loaded with cloxacillin and physico-chemically characterized. MICs were determined for reference strains Newman-(MSSA) and USA300-(MRSA). Murine alveolar macrophages were infected, and bacterial intracellular survival was assessed after incubating with free-cloxacillin or PLGA-cloxacillin-NP., [Results and conclusion]: For both isolates, MICs for antibiotic-loaded-NP were lower than those obtained with free cloxacillin, indicating that the drug encapsulation improves antimicrobial activity. A sustained antibiotic release was demonstrated when using the PLGA-cloxacillin-NP. When considering the lowest concentrations, the use of drug-loaded NP enabled a higher reduction of intracellular bacterial load., This project was funded jointly by SEPAR (Spanish Society for Pneumology and Thoracic Surgery), CIBER-BBN and CIBERES (Spanish Consortia for Research on Bioengineering, Biomaterials and Nanomedicine, and on Respiratory Diseases, respectively). This research was also supported by the Instituto de Salud Carlos III (PI 17/01139), integrated in the Plan Nacional de I+D+I and co-funded jointly by the ISCIII Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER). The authors also thank the support from The Spanish Ministry of Science, Innovation and Universities (grant: CTQ2017-84473-R). We also acknowledge financial support from the EU (ERC Consolidator grant program, ERC-2013-CoG-614715, NANOHEDONISM). J Domínguez and G Mendoza are researchers from the Miguel Servet Program. C Prat was awarded by Programa Germans Trias Sapiens-Fundació Catalunya la Pedrera and European Respiratory Society – ERS Short-Term Research Fellowship 2018 (STRTF201810-00467).

Infectious diseases remain a major burden to global health. Despite the implementation of successful vaccination campaigns and efficient drugs, the increasing emergence of pathogenic vaccine or treatment resistance demands novel therapeutic strategies. The development of traditional therapies using small-molecule drugs is based on modulating protein function and activity through the occupation of active sites such as enzyme inhibition or ligand-receptor binding. These prerequisites result in the majority of host and pathogenic disease-relevant, nonenzymatic and structural proteins being labeled "undruggable." Targeted protein degradation (TPD) emerged as a powerful strategy to eliminate proteins of interest including those of the undruggable variety. Proteolysis-targeting chimeras (PROTACs) are rationally designed heterobifunctional small molecules that exploit the cellular ubiquitin-proteasome system to specifically mediate the highly selective and effective degradation of target proteins. PROTACs have shown remarkable results in the degradation of various cancer-associated proteins, and several candidates are already in clinical development. Significantly, PROTAC-mediated TPD holds great potential for targeting and modulating pathogenic proteins, especially in the face of increasing drug resistance to the best-in-class treatments. In this review, we discuss advances in the development of TPD in the context of targeting the host-pathogen interface and speculate on their potential use to combat viral, bacterial, and parasitic infection.

Rates of antibiotic and multidrug resistance are rapidly rising, leaving fewer options for successful treatment of bacterial infections. In addition to acquiring genetic resistance, many pathogens form persister cells, form biofilms, and/or cause intracellular infections that enable bacteria to withstand antibiotic treatment and serve as a source of recurring infections. JD1 is a small molecule previously shown to kill Gram-negative bacteria under conditions where the outer membrane and/or efflux pumps are disrupted. We show here that JD1 rapidly disrupts membrane potential and kills Gram-positive bacteria. Further investigation revealed that treatment with JD1 disrupts membrane barrier function and causes aberrant membranous structures to form. Additionally, exposure to JD1 reduced the number of Staphylococcus aureus and Staphylococcus epidermidis viable persister cells within broth culture by up to 1,000-fold and reduced the matrix and cell volume of biofilms that had been established for 24 h. Finally, we show that JD1 reduced the number of recoverable methicillin-resistant S. aureus organisms from infected cells. These observations indicate that JD1 inhibits staphylococcal cells in difficult-to-treat growth stages as well as, or better than, current clinical antibiotics. Thus, JD1 shows the importance of testing compounds under conditions that are relevant to infection, demonstrates the utility that membrane-targeting compounds have against multidrug-resistant bacteria, and indicates that small molecules that target bacterial cell membranes may serve as potent broad-spectrum antibacterials. IMPORTANCE Untreatable bacterial infections are a critical public health care issue. In addition to increasing antibiotic resistance, bacteria that are in slow-growing or nongrowing states, or that live inside mammalian cells, are typically insensitive to clinical antibiotics and therefore difficult to eradicate. Bacterial cell membranes have been proposed as potential novel antibiotic targets that may be vulnerable in these difficult to treat cell types because cell membranes are always present and performing essential functions. The small molecule JD1 was previously shown to disrupt Gram-negative bacterial cell membranes. Here, we show that it also disrupts the cell membrane of Gram-positive bacteria and reduces viable bacteria within persister populations, biofilms, and mammalian cells. These observations demonstrate the importance of testing novel compounds under infection-relevant conditions, because their potency against rapidly growing cells may not reveal their full potential.

The development of antibiotic resistance in bacteria is a major public health threat. Infection rates of resistant pathogens continue to rise against nearly all antimicrobials, which has led to development of different strategies to combat the antimicrobial resistance. In this review, we discuss how the newly popular CRISPR-cas system has been applied to combat antibiotic resistance in both extracellular and intracellular pathogens. We also review a recently developed method in which nano-size CRISPR complex was used without any phage to target the mecA gene. However, there is still challenge to practice these methods in field against emerging antimicrobial resistant pathogens.

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized, however, little is known about the &gt, 25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

Background Chronic infection by Staphylococcus aureus drives pathogenesis in important clinical settings, such as recurrent pulmonary infection in cystic fibrosis and relapsing infection in osteomyelitis. Treatment options for intracellular S. aureus infection are limited. Rifampin, a lipophilic antibiotic, readily penetrates host cell membranes, yet monotherapy is associated with rapid antibiotic resistance and development of severe adverse events. Antibiotic cotreatment can reduce this progression, yet efficacy diminishes as antibiotic resistance develops. ML141 and simvastatin inhibit S. aureus invasion through host-directed rather than bactericidal mechanisms. Objective To determine whether cotreatment of ML141 or of simvastatin with rifampin would enhance rifampin efficacy. Methods Assays to assess host cell invasion, host cell viability, host cell membrane permeability, and bactericidal activity were performed using the human embryonic kidney (HEK) 293-A cell line infected with S. aureus (29213) and treated with vehicle control, simvastatin, ML141, rifampin, or cotreatment of simvastatin or ML141 with rifampin. Results We found cotreatment of ML141 with rifampin reduced intracellular infection nearly 85% when compared to the no treatment control. This decrease more than doubled the average 40% reduction in response to rifampin monotherapy. In contrast, cotreatment of simvastatin with rifampin failed to improve rifampin efficacy. Also, in contrast to ML141, simvastatin increased propidium iodide (PI) positive cells, from an average of 10% in control HEK 293-A cells to nearly 20% in simvastatin-treated cells, indicating an increase in host cell membrane permeability. The simvastatin-induced increase was reversed to control levels by cotreatment of simvastatin with rifampin. Conclusion Taken together, rifampin efficacy is increased through host-directed inhibition of S. aureus invasion by ML141, while efficacy is not increased by simvastatin. Considerations regarding novel therapeutic approaches may be dependent on underlying differences in pharmacology.

Thiol-disulfide glutaredoxin systems of bacterial cytoplasm favor reducing conditions for the correct disulfide bonding of functional proteins, and therefore were employed by bacteria to defend against oxidative stress. Listeria monocytogenes has been shown to encode a putative glutaredoxin, Grx (encoded by lmo2344), while the underlying roles remain unknown. Here we suggest an unexpected role of L. monocytogenes Grx in oxidative tolerance and intracellular infection. The recombinant Grx was able to efficiently catalyze the thiol-disulfide oxidoreduction of insulin in the presence of DTT as an election donor. Unexpectedly, the deletion of grx resulted in a remarkably increased tolerance and survival ability of this bacteria when exposed to various oxidizing agents, including diamide, and copper and cadmium ions. Furthermore, loss of grx significantly promoted bacterial invasion and proliferation in human epithelial Caco-2 cells and murine macrophages, as well as a notably increasing invasion but not cell-to-cell spread in the murine fibroblasts L929 cells. More importantly, L. monocytogenes lacking the glutaredoxin exhibited more efficient proliferation and recovery in the spleens and livers of the infected mice, and hence became more virulent by upregulating the virulence factors, InlA and InlB. In summary, we here for the first time demonstrated that L. monocytogenes glutaredoxin plays a counterintuitive role in bacterial oxidative resistance and intracellular infection, which is the first report to provide valuable evidence for the role of glutaredoxins in bacterial infection, and more importantly suggests a favorable model to illustrate the functional diversity of bacterial Grx systems during environmental adaption and host infection.

Wolbachia, Rickettsia, Spiroplasma and Cardinium are endosymbiotic and intracellular bacteria known to cause numerous disorders in host reproduction, reflected in their common name “male-killers”. In this study, 297 beetle species from various taxonomic groups were screened with the use of molecular markers for the presence of infection by any of these endosymbionts. Wolbachia was found to be the most common “male-killer” among beetle hosts as it infected approx. 27% of species. Rickettsia, Spiroplasma and Cardinium were much less prevalent as they infected: 8%, 3% and 2%, respectively, of the studied beetle species. This is the first report of Cardinium presence in beetle hosts. Incidences of co-infection of two bacteria taxa were very rare and only two weevil species were found to be infected by three different bacteria. These findings suggest that endosymbiotic bacteria inhabit their host at various levels of prevalence and that particular taxa usually infect different hosts, suggesting some competition among “male-killers”. This is the first study that simultaneously verifies infection status by all major endosymbiotic bacteria in hundreds of species (based on coleopterans).

The promising antibacterial potency of arylthiazole antibiotics is offset by their limited activity against intracellular bacteria (namely methicillin-resistant Staphylococcus aureus (MRSA)), similar to many clinically-approved antibiotics. The failure to target these hidden pathogens is due to the compounds' lack of proper characteristics to accumulate intracellularly. Fine tuning of the size and polar-surface-area of the linking heteroaromatic ring provided a new series of 5-thiazolylarylthiazoles with balanced properties that allow them to sufficiently cross and accumulate inside macrophages infected with MRSA. The most promising compound 4i exhibited rapid bactericidal activity, good metabolic stability and produced over 80% reduction of intracellular MRSA in infected macrophages.

In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.

Background Porphyromonas gingivalis possess the ability to invade host cells which prevents this pathogen from eradication by conventional periodontal therapy. Recently, antimicrobial photodynamic therapy (aPDT) was introduced to periodontal treatment as a complementary antibacterial method. The aim of this study was to evaluate the effect of toluidine blue-O (TBO) mediated aPDT on the viability of P. gingivalis invading gingival fibroblasts and keratinocytes in an in vitro model of infection. Methods Primary human gingival fibroblasts (PHGF) and telomerase immortalized gingival keratinocytes (TIGK) were infected with Pg ATCC 33277. Two concentrations of TBO (0.01 mg/mL, TBO-c1 and 0.001 mg/mL, TBO-c2) and a non-laser red light source (λ = 630 nm) were applied to treat both cell-adherent/intracellular Pg (the adhesion/invasion model) or exclusively the intracellular bacteria (the intracellular infection model). Results The median viability of cell-adherent/intracellular Pg in infected keratinocytes declined from 1.88 × 105 cfu/mL in infected cells treated with TBO without irradiation to 40 cfu/mL upon irradiation for 10 s with TBO-c1. At higher light doses a complete photokilling of P. gingivalis was observed. Pg from exclusively intracellular infection model was also efficiently eradicated as the residual viability dropped from 1.44 × 105 cfu/mL in control samples to 160, 20 and 10 cfu/mL upon irradiation for 10, 20 and 30 s, respectively. In the infected fibroblasts irradiation significantly reduced bacterial viability but did not completely eradicate the intracellular pathogen. Conclusions Antimicrobial PDT is effective in reducing the viability of intracellular periopathogens, however those residing within gingival fibroblasts seems to attenuate the photokilling effectiveness of this method.

Francisella tularensis is a highly infectious intracellular pathogen that infects a wide range of host species and causes fatal pneumonic tularemia in humans. ftlA was identified as a potential virulence determinant of the F. tularensis live vaccine strain (LVS) in our previous transposon screen, but its function remained undefined. Here, we show that an unmarked deletion mutant of ftlA was avirulent in a pneumonia mouse model with a severely impaired capacity to infect host cells. Consistent with its sequence homology with GDSL lipase/esterase family proteins, the FtlA protein displayed lipolytic activity in both E. coli and F. tularensis with a preference for relatively short carbon-chain substrates. FtlA thus represents the first F. tularensis lipase to promote bacterial infection of host cells and in vivo fitness. As a cytoplasmic protein, we found that FtlA was secreted into the extracellular environment as a component of outer membrane vesicles (OMVs). Further confocal microscopy analysis revealed that the FtlA-containing OMVs isolated from F. tularensis LVS attached to the host cell membrane. Finally, the OMV-associated FtlA protein complemented the genetic deficiency of the ΔftlA mutant in terms of host cell infection when OMVs purified from the parent strain were co-incubated with the mutant bacteria. These lines of evidence strongly suggest that the FtlA lipase promotes F. tularensis adhesion and internalization by modifying bacterial and/or host molecule(s) when it is secreted as a component of OMVs.

Persisters are the fraction of antibiotic-exposed bacteria transiently refractory to killing and are recognized as a cause of antibiotic treatment failure. The putative de-N-acetylase DnpA increases persister levels in Pseudomonas aeruginosa upon exposure to fluoroquinolones in broth. In this study the wild-type PAO1 and its dnpA insertion mutant (dnpA::Tn) were used in parallel and compared for their capacity to generate persisters in broth (surviving fraction after exposure to high antibiotic concentrations) and their susceptibility to antibiotics in models of intracellular infection of THP-1 monocytes and of biofilms grown in microtiter plates. Multiplication in monocytes was evaluated by fluorescence dilution of GFP (expressed under the control of an inducible promoter) using flow cytometry. Gene expression was measured by quantitative RT-PCR. When exposed to fluoroquinolones (ciprofloxacin or levofloxacin) but not to meropenem or amikacin, the dnpA::Tn mutant showed a 3- to 10-fold lower persister fraction in broth. In infected monocytes, fluoroquinolones (but not the other antibiotics) were more effective (difference in Emax: 1.5 log cfu) against the dnpA::Tn mutant than against the wild-type PAO1. Dividing intracellular bacteria were more frequently seen (1.5 to 1.9-fold) with the fluoroquinolone-exposed dnpA::Tn mutant than with its parental strain. Fluoroquinolones (but not the other antibiotics) were also 3-fold more potent to prevent biofilm formation by the dnpA::Tn mutant than by PAO1 as well as to act upon biofilms (1-3 days of maturity) formed by the mutant than by the parental strain. Fluoroquinolones induced the expression of gyrA (4.5-7 fold) and mexX (3.6-5.4 fold) in the parental strain but to a lower extent (3-4-fold for gyrA and 1.8-2.8-fold for mexX, respectively) in the dnpA::Tn mutant. Thus, our data show that a dnpA insertion mutant of P. aeruginosa is more receptive to fluoroquinolone antibacterial effects than its parental strain in infected monocytes or in biofilms. The mechanism of this higher responsiveness could involve a reduced overexpression of the fluoroquinolone target. ispartof: FRONTIERS IN MICROBIOLOGY vol:9 issue:JUL ispartof: location:Switzerland status: published

Legionella pneumophila genes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection of Acanthamoeba castellanii, although these genes had been considered dispensable on the basis of the behavior of mutants lacking either lapA and lapB or plaC. A plaC mutant showed even higher levels of lapA and lapB transcripts, and a lapA lapB mutant showed heightening of plaC mRNA levels, suggesting that the role of the LapA/B aminopeptidase is compensatory with respect to that of the PlaC acyltransferase. Hence, we made double mutants and found that lapA plaC mutants have an ~50-fold defect during infection of A. castellanii. These data revealed, for the first time, the importance of LapA in any sort of infection; thus, we purified LapA and defined its crystal structure, activation by another T2S-dependent protease (ProA), and broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of the lapA plaC mutant was reversed, implying that LapA generates amino acids for nutrition. Since the LapA and PlaC data did not fully explain the role of T2S in infection, we identified, via proteomic analysis, a novel secreted protein (NttD) that promotes infection of A. castellanii. A lapA plaC nttD mutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data explain, to a significant degree, the importance of T2S. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside the Legionella genus. LapA was notable for having as its closest homologue an A. castellanii protein., IMPORTANCE Transmission of L. pneumophila to humans is facilitated by its ability to grow in Acanthamoeba species. We previously documented that type II secretion (T2S) promotes L. pneumophila infection of A. castellanii. Utilizing transcriptional analysis and proteomics, double and triple mutants, and crystal structures, we defined three secreted substrates/effectors that largely clarify the role of T2S during infection of A. castellanii. Particularly interesting are the unique functional overlap between an acyltransferase (PlaC) and aminopeptidase (LapA), the broad substrate specificity and eukaryotic-protein-like character of LapA, and the novelty of NttD. Linking LapA to amino acid acquisition, we defined, for the first time, the importance of secreted aminopeptidases in intracellular infection. Bioinformatic investigation, not previously applied to T2S, revealed that effectors originate from diverse sources and distribute within the Legionella genus in unique ways. The results of this study represent a major advance in understanding Legionella ecology and pathogenesis, bacterial secretion, and the evolution of intracellular parasitism.

Streptococcus pyogenes (Group A Streptococcus; GAS) commonly causes pharyngitis in children and adults, with severe invasive disease and immune sequelae being an infrequent consequence. The ability of GAS to invade the host and establish infection likely involves subversion of host immune defenses. However, the signaling pathways and innate immune responses of epithelial cells to GAS are not well-understood. In this study, we utilized RNAseq to characterize the inflammatory responses of primary human tonsil epithelial (TEpi) cells to infection with the laboratory-adapted M6 strain JRS4 and the M1T1 clinical isolate 5448. Both strains induced the expression of genes encoding a wide range of inflammatory mediators, including IL-8. Pathway analysis revealed differentially expressed genes between mock and JRS4- or 5448-infected TEpi cells were enriched in transcription factor networks that regulate IL-8 expression, such as AP-1, ATF-2, and NFAT. While JRS4 infection resulted in high levels of secreted IL-8, 5448 infection did not, suggesting that 5448 may post-transcriptionally dampen IL-8 production. Infection with 5448ΔcepA, an isogenic mutant lacking the IL-8 protease SpyCEP, resulted in IL-8 secretion levels comparable to JRS4 infection. Complementation of 5448ΔcepA and JRS4 with a plasmid encoding 5448-derived SpyCEP significantly reduced IL-8 secretion by TEpi cells. Our results suggest that intracellular infection with the pathogenic GAS M1T1 clone induces a strong pro-inflammatory response in primary tonsil epithelial cells, but modulates this host response by selectively degrading the neutrophil-recruiting chemokine IL-8 to benefit infection.

Eukaryotic proteome diversity exceeds that encoded within individual genes, and results in part from alternative splicing events of pre-messenger RNA. The diversity of these splicing events can shape the outcome in development and differentiation of normal tissues, and is important in pathogenic circumstances such as cancer and some heritable conditions. A role for alternative splicing of eukaryotic genes in response to viral and intracellular bacterial infections has only recently been recognized, and plays an important role in providing fitness for microbial survival, while potentially enhancing pathogenicity. Anaplasma phagocytophilum survives within mammalian neutrophils by reshaping transcriptional programs that govern cellular functions. We applied next generation RNAseq to ATRA-differentiated HL-60 cells established to possess transcriptional and functional responses similar to A. phagocytophilum-infected human neutrophils. This demonstrated an increase in transcripts with infection and high proportion of alternatively spliced transcript events (ASEs) for which predicted gene ontology processes were in part distinct from those identified by evaluation of single transcripts or gene-level analyses alone. The alternative isoforms are not on average shorter, and no alternative splicing in genes encoding spliceosome components is noted. Although not evident at gene-level analyses, individual spliceosome transcripts that impact nearly all spliceosome components were significantly upregulated. How the distinct GO processes predicted by ASEs are regulated by infection and whether they are relevant to fitness or pathogenicity of A. phagocytophilum should be addressed in more detailed studies.

Brucellae are facultative intracellular pathogens. The recent development of methods and genetically engineered strains allowed the description of cell-cycle progression of. Brucella abortus, including unipolar growth and the ordered initiation of chromosomal replication. B. abortus cell-cycle progression is coordinated with intracellular trafficking in the endosomal compartments. Bacteria are first blocked at the G1 stage, growth and chromosome replication being resumed shortly before reaching the intracellular proliferation compartment. The control mechanisms of cell cycle are similar to those reported for the bacterium. Caulobacter crescentus, and they are crucial for survival in the host cell. The development of single-cell analyses could also be applied to other bacterial pathogens to investigate their cell-cycle progression during infection. Brucella abortus is a facultative intracellular pathogen that grows unipolarly and initiates the replication of its two chromosomes in a specific order.Bacteria at the G1 stage of the cell cycle, that is, before the initiation of their chromosomal replication, are preferentially internalized in host cells.Cell-cycle progression is coordinated with trafficking in the host cell, the endosomal stage being divided into two parts: a first, long, nongrowing part, and a second part in which growth and chromosomal replication are resumed.A cell-cycle control network, conserved with the model bacterium. Caulobacter crescentus, is essential for. B. abortus virulence.

The complement system is an ancient part of the innate immune system important for both tissue homeostasis and host defense. However, bacteria like Staphylococcus aureus (SA) possess elaborative mechanisms for evading both the complement system and other parts of the immune system. One of these evasive mechanisms-important in causing chronic and therapy resistant infections-is the intracellular persistence in non-immune cells. The objective of our study was to investigate whether persistent intracellular SA infection of epidermal keratinocytes resulted in complement activation. Using fluorescence microscopy, we found that persistent SA, surviving intracellularly in keratinocytes, caused activation of the complement system with formation of the terminal complement complex (TCC) at the cell surface. Skin samples from atopic dermatitis patients analyzed by bacterial culture and microscopy, demonstrated that SA colonization was associated with the presence of intracellular bacteria and deposition of the TCC in epidermis in vivo. Complement activation on keratinocytes with persistent intracellular bacteria was found with sera deficient/depleted of the complement components C1q, Mannan-binding lectin, or complement factor B, demonstrating involvement of more than one complement activation pathway. Viable bacterial counts showed that complement activation at the cell surface initiated cellular responses that significantly reduced the intracellular bacterial burden. The use of an inhibitor of the extracellular signal-regulated kinase (ERK) abrogated the complement-induced reduction in intracellular bacterial load. These data bridge the roles of the complement system in tissue homeostasis and innate immunity and illustrate a novel mechanism by which the complement system combats persistent intracellular bacteria in epithelial cells.

Xiaolin Zhang,1,2,* Jiankang Song,3,* Alexey Klymov,3,* Yang Zhang,3 Leonie de Boer,1 John A Jansen,3 Jeroen JJP van den Beucken,3 Fang Yang,3 Sebastian AJ Zaat,1,* Sander CG Leeuwenburgh3,* 1Department of Medical Microbiology, Amsterdam Infection and Immunity Institute, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; 2Department of Biomaterials Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, the Netherlands; 3Department of Biomaterials, Radboud University Medical Centre, Nijmegen, the Netherlands *These authors contributed equally to this work Background: Infections such as biomaterial-associated infection and osteomyelitis are often associated with intracellular survival of bacteria (eg, Staphylococcus aureus). Treatment of these infections remains a major challenge due to the low intracellular efficacy of many antibiotics. Therefore, local delivery systems are urgently required to improve the therapeutic efficacy of antibiotics by enabling their intracellular delivery. Purpose: To assess the potential of gelatin nanospheres as carriers for local delivery of vancomycin into macrophages of zebrafish larvae in vivo and into THP-1-derived macrophages in vitro using fluorescence microscopy. Materials and methods: Fluorescently labeled gelatin nanospheres were prepared and injected into transgenic zebrafish larvae with fluorescent macrophages. Both the biodistribution of gelatin nanospheres in zebrafish larvae and the co-localization of vancomycin-loaded gelatin nanospheres with zebrafish macrophages in vivo and uptake by THP-1-derived macrophages in vitro were studied. In addition, the effect of treatment with vancomycin-loaded gelatin nanospheres on survival of S. aureus-infected zebrafish larvae was investigated. Results: Internalization of vancomycin-loaded gelatin nanospheres by macrophages was observed qualitatively both in vivo and in vitro. Systemically delivered vancomycin, on the other hand, was hardly internalized by macrophages without the use of gelatin nanospheres. Treatment with a single dose of vancomycin-loaded gelatin nanospheres delayed the mortality of S. aureus-infected zebrafish larvae, indicating the improved therapeutic efficacy of vancomycin against (intracellular) S. aureus infection in vivo. Conclusion: The present study demonstrates that gelatin nanospheres can be used to facilitate local and intracellular delivery of vancomycin. Keywords: in vivo real-time monitoring, fluorescence microscopy, biodistribution, cell-material interaction, Staphylococcus aureus, intracellular infection

As a facultative intracellular pathogen, Staphylococcus aureus is able to invade and proliferate within many types of mammalian cells. Intracellular bacterial replication relies on host nutrient supplies and, therefore, cell metabolism is closely bound to intracellular infection. Here, we investigated how S. aureus invasion affects the host membrane-bound fatty acids. We quantified the relative levels of fatty acids and their labelling pattern after intracellular infection by gas chromatography-mass spectrometry (GC-MS). Interestingly, we observed that the levels of three host fatty acids—docosanoic, eicosanoic and palmitic acids—were significantly increased in response to intracellular S. aureus infection. Accordingly, labelling carbon distribution was also affected in infected cells, in comparison to the uninfected control. In addition, treatment of HeLa cells with these three fatty acids showed a cytoprotective role by directly reducing S. aureus growth.

The use of nanoparticle delivery systems to enhance intracellular penetration of antibiotics and their retention time is becoming popular. The challenge, however, is that the interaction of nanoparticles with biological systems at the cellular level must be established prior to biomedical applications. Ciprofloxacin–cockle shells-derived calcium carbonate (aragonite) nanoparticles (C-CSCCAN) were developed and characterized. Antibacterial activity was determined using a modified disc diffusion protocol on Salmonella Typhimurium (S. Typhimurium). Biocompatibilittes with macrophage were evaluated using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Bromo-2′-deoxyuridine (BrdU) assays. Transcriptional regulation of interleukin 1 beta (IL-1β) was determined using reverse transcriptase-polymerase chain reaction (RT-PCR). C-CSCCAN were spherical in shape, with particle sizes ranging from 11.93 to 22.12 nm. Encapsulation efficiency (EE) and loading content (LC) were 99.5% and 5.9%, respectively, with negative ζ potential. X-ray diffraction patterns revealed strong crystallizations and purity in the formulations. The mean diameter of inhibition zone was 18.6 ± 0.5 mm, which was better than ciprofloxacin alone (11.7 ± 0.9 mm). Study of biocompatability established the cytocompatability of the delivery system without upregulation of IL-1β. The results indicated that ciprofloxacin–nanoparticles enhanced the antibacterial efficacy of the antibiotic, and could act as a suitable delivery system against intracellular infections.

Francisella tularensis is a highly virulent facultative intracellular bacterium. The lack of a safe and efficient vaccine makes antibiotics the preferred treatment. F. tularensis antibiotic susceptibility tests are based on the in vitro standard CLSI-approved microdilution method for determining the MIC. However, limited data are available regarding the minimal inhibitory extracellular concentration (MIEC) needed to eradicate intracellular bacteria. Here, we evaluated the MIEC values of various WHO-recommended antibiotics and compared the MIEC values to the established MICs. We describe a rapid 3-h quantitative PCR (qPCR) intracellular antibiogram assay, which yields comparable MIEC values to those obtained by the classical 72-h cfu assay. This rapid qPCR assay is highly advantageous in light of the slow growth rates of F. tularensis. Our results showed that the MIECs obtained for doxycycline, chloramphenicol and ciprofloxacin were indicative of intracellular activity. Gentamicin was not effective against intracellular bacteria for at least 32 h post treatment, raising the question of whether slow-penetrating gentamicin should be used for certain stages of the disease. We suggest that the qPCR intracellular antibiogram assay may be used to screen for potentially active antibiotics against intracellular F. tularensis as well as to detect strains with acquired resistance to recommended antibiotics.

Computational models can provide valuable insights into the mechanisms of infection and be used as investigative tools to support development of medical treatments. We develop a stochastic, within-host, computational model of the infection process in the BALB/c mouse, following inhalational exposure to Francisella tularensis SCHU S4. The model is mechanistic and governed by a small number of experimentally verifiable parameters. Given an initial dose, the model generates bacterial load profiles corresponding to those produced experimentally, with a doubling time of approximately 5 hours during the first 48 hours of infection. Analytical approximations for the mean number of bacteria in phagosomes and cytosols for the first twenty-four hours post-infection are derived and used to verify the stochastic model. In our description of the dynamics of macrophage infection, the number of bacteria released per rupturing macrophage is a geometrically-distributed random variable. When combined with doubling time, this provides a distribution for the time taken for infected macrophages to rupture and release their intracellular bacteria. The mean and variance of these distributions are determined by model parameters with a precise biological interpretation, providing new mechanistic insights into the determinants of immune and bacterial kinetics. Insights into the dynamics of macrophage suppression and activation gained by the model can be used to explore the potential benefits of interventions that stimulate macrophage activation.

Recently, we have reported the presence of fungal infections in patients with Alzheimer’s disease (AD). Accordingly, fungal proteins and DNA were found in brain samples, demonstrating the existence of infection in the central nervous system. In the present work, we raised antibodies to specific fungal species and performed immunohistochemistry to directly visualize fungal components inside neurons from AD patients. Mice infected with Candida glabrata were initially used to assess whether yeast can be internalized in mammalian tissues. Using polyclonal rabbit antibodies against C. glabrata, rounded immunopositive cells could be detected in the cytoplasm of cells from liver, spleen, and brain samples in infected, but not uninfected, mice.Immunohistochemical analyses of tissue from the frontal cortex of AD patients revealed the presence of fungal material in a small percentage (∼10%) of cells, suggesting the presence of infection. Importantly, this immunopositive material was absent in control samples. Confocal microscopy indicated that this fungal material had an intracellular localization. The specific morphology of this material varied between patients; in some instances, disseminated material was localized to the cytoplasm, whereas small punctate bodies were detected in other patients. Interestingly, fungal material could be revealed using different anti-fungal antibodies, suggesting multiple infections. In summary, fungal infection can only be observed using specific antifungal antibodies and only a small percentage of cells contain fungi. Our findings provide an explanation for the hitherto elusive detection of fungi in AD brains, and are consistent with the idea that fungal cells are internalized inside neurons., Fundación ONCE (Organización Nacional de Ciegos Espa˜noles) is acknowledged.We acknowledge an institutional grant to Centro de Biología Molecular “Severo Ochoa” from the Fundación Ramón Areces.

In a previous study (L. G. Garcia et al., Antimicrob. Agents Chemother. 56:3700–3711, 2012), we evaluated the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent small-colony variants, respectively) of the parental COL methicillin-resistant Staphylococcus aureus strain and the pharmacodynamic profile of the intracellular activity of a series of antibiotics in human THP-1 monocytes. We have now examined the phagocytosis and intracellular persistence of the same strains in THP-1 cells activated by phorbol 12-myristate 13-acetate (PMA) and measured the intracellular activity of gentamicin, moxifloxacin, and oritavancin in these cells. Postphagocytosis intracellular counts and intracellular survival were lower in PMA-activated cells, probably due to their higher killing capacities. Gentamicin and moxifloxacin showed a 5- to 7-fold higher potency (lower static concentrations) against the parental strain, its hemB mutant, and the genetically complemented strain in PMA-activated cells and against the menD strain in both activated and nonactivated cells. This effect was inhibited when cells were incubated with N -acetylcysteine (a scavenger of oxidant species). In parallel, we observed that the MICs of these drugs were markedly reduced if bacteria had been preexposed to H 2 O 2 . In contrast, the intracellular potency of oritavancin was not different in activated and nonactivated cells and was not decreased by the addition of N -acetylcysteine, regardless of the phenotype of the strains. The oritavancin MIC was also unaffected by preincubation of the bacteria with H 2 O 2 . Thus, activation of THP-1 cells by PMA may increase the intracellular potency of certain antibiotics (probably due to synergy with reactive oxygen species), but this effect cannot be generalized to all antibiotics.

Background Bacteria which are metabolically active yet unable to be cultured and eradicated by antibiotic treatment are present in the middle ear effusion of children with chronic otitis media with effusion (COME) and recurrent acute otitis media (rAOM). These observations are suggestive of biofilm presence or intracellular sequestration of bacteria and may play a role in OM pathogenesis. The aim of this project is to provide evidence for the presence of otopathogenic bacteria intracellularly or within biofilm in the middle ear mucosa of children with COME or rAOM. Methods Middle ear mucosal biopsies from 20 children with COME or rAOM were examined for otopathogenic bacteria (either in biofilm or located intracellularly) using transmission electron microscopy (TEM) or species specific fluorescent in situ hybridisation (FISH) and confocal laser scanning microscopy (CLSM). One healthy control biopsy from a child undergoing cochlear implant surgery was also examined. Results No bacteria were observed in the healthy control sample. In 2 of the 3 biopsies imaged using TEM, bacteria were observed in mucus containing vacuoles within epithelial cells. Bacterial species within these could not be identified and biofilm was not observed. Using FISH with CLSM, bacteria were seen in 15 of the 17 otitis media mucosal specimens. In this group, 11 (65%) of the 17 middle ear mucosal biopsies showed evidence of bacterial biofilm and 12 demonstrated intracellular bacteria. 52% of biopsies were positive for both biofilm and intracellular bacteria. At least one otopathogen was identified in 13 of the 15 samples where bacteria were present. No differences were observed between biopsies from children with COME and those with rAOM. Conclusion Using FISH and CLSM, bacterial biofilm and intracellular infection with known otopathogens are demonstrated on/in the middle ear mucosa of children with COME and/or rAOM. While their role in disease pathogenesis remains to be determined, this previously undescribed infection pattern may help explain the ineffectiveness of current treatment strategies at preventing or resolving COME or rAOM.