Intracellular Bacterial Survival Research Articles

Roles of Critical Amino Acids Metabolism in The Interactions Between Intracellular Bacterial Infection and Macrophage Function.

Macrophages, as crucial participants in the innate immune system, respond to pathogenic challenges through their dynamic metabolic adjustments, demonstrating the intimate interplay between cellular metabolism and immune function. Bacterial infection of macrophages causes changes in macrophage metabolism, affecting both macrophage function and bacterial virulence and intracellular survival. This review explores the reprogramming of amino acid metabolism in macrophages in response to bacterial infection, with a particular focus on the influence of critical amino acids such as serine, glutamine, and arginine on the immune functions of macrophages; highlights the roles of these metabolic pathways in macrophage functions such as phagocytosis, inflammatory response, immune regulation, and pathogen clearance; reveals how pathogens exploit and manipulate the amino acid metabolism within macrophages to support their own growth and replication, thereby showcasing the intricate interplay between macrophages and pathogens. It provides a foundation for understanding the interactions between macrophages amino acid metabolism and pathogens, offering potential strategies and therapeutic targets for the development of novel anti-infection therapies.

Bordetella pertussis outer membrane vesicles impair neutrophil bactericidal activity

Neutrophils constitute the primary defense against bacterial infections, yet certain pathogens express virulence factors that enable them to subvert neutrophils-mediated killing. Outer membrane vesicles (OMVs) have emerged as a secretory system through which bacteria deliver virulence factors to host cells. OMVs from Bordetella pertussis, the etiological agent of whooping cough, are loaded with most of bacterial virulence factors, including CyaA, which plays a key role in B. pertussis evasion of neutrophils bactericidal activity. In our study, we investigated the role of B. pertussis OMVs in bacterial interaction with neutrophils. We observed that interaction of OMVs with neutrophils led to a decrease in the expression of cell surface CR3 and FcγRs, an effect dependent on the CyaA toxin delivered by these vesicles. This decreased receptor expression led to reduced bacterial uptake by neutrophils, irrespective of the presence of opsonic antibodies. Moreover, CyaA delivered by OMVs hindered intracellular bactericidal trafficking, promoting bacterial intracellular survival. When both bacteria and OMVs were opsonized, competition between opsonized OMVs and B. pertussis for FcγRs on neutrophils led to a significant decrease in bacterial uptake. Overall, our findings suggest that B. pertussis OMVs promote bacterial survival to the encounter with neutrophils in both naïve and immunized individuals.

Function of Burkholderia pseudomallei RpoS and RpoN2 in bacterial invasion, intracellular survival, and multinucleated giant cell formation in mouse macrophage cell line.

Melioidosis, a human infectious disease with a high mortality rate in many tropical countries, is caused by the pathogen Burkholderia pseudomallei (B. pseudomallei). The function of the B. pseudomallei sigma S (RpoS) transcription factor in survival during the stationary growth phase and conditions of oxidative stress is well documented. Besides the rpoS, bioinformatics analysis of B. pseudomallei genome showed the existence of two rpoN genes, named rpoN1 and rpoN2. In this study, by using the mouse macrophage cell line RAW264.7 as a model of infection, the involvement of B. pseudomallei RpoS and RpoN2 in the invasion, intracellular survival leading to the reduction in multinucleated giant cell (MNGC) formation of RAW264.7 cell line were illustrated. We have demonstrated that the MNGC formation of RAW264.7 cell was dependent on a certain number of intracellular bacteria (at least 5 × 104). In addition, the same MNGC formation (15%) observed in RAW264.7 cells infected with either B. pseudomallei wild type with multiplicity of infection (MOI) 2 or RpoN2 mutant (∆rpoN2) with MOI 10 or RpoS mutant (∆rpoS) with MOI 100. The role of B. pseudomallei RpoS and RpoN2 in the regulation of type III secretion system on bipB-bipC gene expression was also illustrated in this study.

A genome-wide CRISPR screen identified host genes essential for intracellular Brucella survival.

Brucella is a zoonotic intracellular bacterium that poses threats to human health and economic security. Intracellular infection is a hallmark of the agent Brucella and a primary cause of distress, through which the bacterium regulates the host intracellular environment to promote its own colonization and replication, evading host immunity and pharmaceutical killing. Current studies of Brucella intracellular processes are typically premised on bacterial phenotype such as intracellular bacterial survival, followed by biochemical or molecular biological approaches to reveal detailed mechanisms. While such processes can deepen the understanding of Brucella-host interaction, the insights into host alterations in infection would be easily restricted to known pathways. In the current study, we applied CRISPR Cas9 screen to identify host genes that are most affected by Brucella infection on cell viability at the genomic level. As a result of CRISPR screening, we firstly identified that knockout of the negatively selected genes GOLGA6L6, DEFB103B, OR4F29, and ERCC6 attenuate the viability of both the host cells and intracellular Brucella, suggesting these genes to be potential therapeutic targets for Brucella control. In particular, knockout of DEFB103B diminished Brucella intracellular survival by altering host cell autophagy. Conversely, knockout of positive screening genes promoted intracellular proliferation of Brucella. In summary, we screened host genes at the genomic level throughout Brucella infection, identified host genes that are previously not recognized to be involved in Brucella infection, and provided targets for intracellular infection control.IMPORTANCEBrucella is a Gram-negative bacterium that infects common mammals causing arthritis, myalgia, neuritis, orchitis, or miscarriage and is difficult to cure with antibiotics due to its intracellular parasitism. Therefore, unraveling the mechanism of Brucella-host interactions will help controlling Brucella infections. CRISPR-Cas9 is a gene editing technology that directs knockout of individual target genes by guided RNA, from which genome-wide gene-knockout cell libraries can be constructed. Upon infection with Brucella, the cell library would show differences in viability as a result of the knockout and specific genes could be revealed by genomic DNA sequencing. As a result, genes affecting cell viability during Brucella infection were identified. Further testing of gene function may reveal the mechanisms of Brucella-host interactions, thereby contributing to clinical therapy.

Akkermansia muciniphila-induced trained immune phenotype increases bacterial intracellular survival and attenuates inflammation

The initial exposure to pathogens and commensals confers innate immune cells the capacity to respond distinctively upon a second stimulus. This training capacity might play key functions in developing an adequate innate immune response to the continuous exposure to bacteria. However, the mechanisms involved in induction of trained immunity by commensals remain mostly unexplored. A. muciniphila represents an attractive candidate to study the promotion of these long-term responses. Here, we show that priming of macrophages with live A. muciniphila enhances bacterial intracellular survival and decreases the release of pro- and anti-inflammatory signals, lowering the production of TNF and IL-10. Global transcriptional analysis of macrophages after a secondary exposure to the bacteria showed the transcriptional rearrangement underpinning the phenotype observed compared to acutely exposed cells, with the increased expression of genes related to phagocytic capacity and those involved in the metabolic adjustment conducing to innate immune training. Accordingly, key genes related to bacterial killing and pro-inflammatory pathways were downregulated. These data demonstrate the importance of specific bacterial members in the modulation of local long-term innate immune responses, broadening our knowledge of the association between gut microbiome commensals and trained immunity as well as the anti-inflammatory probiotic potential of A. muciniphila.

Protozoan predation enhances stress resistance and antibiotic tolerance in Burkholderia cenocepacia by triggering the SOS response.

Bacterivorous protists are thought to serve as training grounds for bacterial pathogens by subjecting them to the same hostile conditions that they will encounter in the human host. Bacteria that survive intracellular digestion exhibit enhanced virulence and stress resistance after successful passage through protozoa but the underlying mechanisms are unknown. Here we show that the opportunistic pathogen Burkholderia cenocepacia survives phagocytosis by ciliates found in domestic and hospital sink drains, and viable bacteria are expelled packaged in respirable membrane vesicles with enhanced resistance to oxidative stress, desiccation, and antibiotics, thereby contributing to pathogen dissemination in the environment. Reactive oxygen species generated within the protozoan phagosome promote the formation of persisters tolerant to ciprofloxacin by activating the bacterial SOS response. In addition, we show that genes encoding antioxidant enzymes are upregulated during passage through ciliates increasing bacterial resistance to oxidative radicals. We prove that suppression of the SOS response impairs bacterial intracellular survival and persister formation within protists. This study highlights the significance of protozoan food vacuoles as niches that foster bacterial adaptation in natural and built environments and suggests that persister switch within phagosomes may be a widespread phenomenon in bacteria surviving intracellular digestion.

2107. Anti-Biofilm Potential of Virulence Factor EsxB of Staphylococcus aureus against its own Sensitive Strain: Molecular Evidences

Abstract Background Staphylococcus aureus express different proteins that modulate intracellular bacterial survival and increase antimicrobial resistance with higher mortality rate. The EsxA and EsxB, substrates of the ESAT-6 like secretion system (Ess) are important during intracellular S.aureus infection. The genes EsxA and EsxB are present in close proximity with 6 other genes, including EsaA, EssA, EsaB, EssB, EssC, and EsaC. Methods EsxB virulence gene from vancomycin resistant S. aureus was cloned and transformed into expression cell line E.coli BL21(DE3) cells under IPTG induction. The expressed proteins were purified using affinity and size exclusion chromatography. Purity and protein structures were checked by using NMR spectroscopy and circular dichroism. Further, purified proteins were evaluated for it's hemolytic and antibiofilm property against S. Aureus (ATCC 6538). Results he purified EsaB protein showed well spread spectra of 1H- NMR spectrum, however, the 15N, 1H-HSQC spectrum showed overlapped peaks or contours in the region of 6.5-10 ppm indicating the absence of well folded 3D conformation in solution state. Circular dichroism showed the presence of beta sheets in structure of EsxB virulence factor. EsxB protein showed antibiofilm activity against S.aureus (ATCC 6538) when treated exogenously. It also showed non hemolytic activity indicating it's non-cytotoxic nature. Conclusion This study explains the evidence that bacterial virulent protein EsxB possess antibiofilm activity against its own sensitive strain. Disclosures All Authors: No reported disclosures

Bone-Adhesive Hydrogel for Effective Inhibition of M. tuberculosis and Osteoblast Regeneration.

Currently, bone tuberculosis (TB) treatment largely involves lifelong drug prescriptions and surgical intervention, resulting in poor quality of life for patients. Therefore, the fabrication of injectable scaffolds to form a solid framework around the defective bone region is gaining importance over the extensive use of antimicrobial inhibitors. Herein, we synthesized a novel bone-adhesive and thermoresponsive hydrogel via conjugation of poly(N-isopropylacrylamide-co-glycidyl methacrylate) (PNIPAM-co-GMA) and cysteine (CYS). Thiolation of the polymer enables chemical cross-linking with the bone glycoprotein, enhancing bone adhesion and permitting control of scaffold retention time. The PNIPAM-co-GMA-CYS hydrogel shows higher cross-linking behavior at 37 °C, forms a strong gel in 260 s, and has 151 kPa adhesion strength on cortical bone. The lead compounds 5-methyl-5H-[1,2,4]triazino[5,6-b]indole-3-thiol (MTIT) and N-tert-butyl-4-methyl-6-(5-methyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)pyrimidin-2-amine (TMTIPA) were identified by a high-throughput screening method. Effective MTIT and TMTIPA are encapsulated in bone-adhesive hydrogel separately, and both have a high release rate above >70% in 180 h. The MTIT- and TMTIPA-loaded PNIPAM-co-GMA-CYS showed an excellent bactericidal effect, reducing the relative intracellular bacterial survival in macrophages. Furthermore, the as-synthesized hydrogel has outstanding mechanical and biocompatibility properties to become a bone-replacing material and provide support to promote bone repair. This work presents a novel bone-adhesive PNIPAM-co-GMA-CYS for the sustained release of lead compounds toward promising alternative bone TB treatment.

LuxS contributes to intramacrophage survival of Streptococcus agalactiae by positively affecting the expression of fruRKI operon

The LuxS quorum sensing system is a widespread system employed by many bacteria for cell-to-cell communication. The luxS gene has been demonstrated to play a crucial role in intramacrophage survival of piscine Streptococcus agalactiae, but the underlying mechanism remains largely unknown. In this study, transcriptome analysis, followed by the luxS gene deletion and subsequent functional studies, confirmed that impaired bacterial survival inside macrophages due to the inactivation of luxS was associated with reduced transcription of the fruRKI operon, encoding the fructose-specific phosphotransferase system. Further, luxS was determined not to enhance the transcription of fruRKI operon by binding its promoter, but to upregulate the expression of this operon via affecting the binding ability of catabolite control protein A (CcpA) to the catabolite responsive element (cre) in the promoter of fruRKI. Collectively, our study identifies a novel and previously unappreciated role for luxS in bacterial intracellular survival, which may give a more thorough understanding of the immune evasion mechanism in S. agalactiae.

Liver X Receptor LXRα Promotes Grass Carp Reovirus Infection by Attenuating IRF3-CBP Interaction and Inhibiting RLR Antiviral Signaling.

Liver X receptors (LXRs) are nuclear receptors involved in metabolism and the immune response. Different from mammalian LXRs, which include two isoforms, LXRα and LXRβ, only a single LXRα gene exists in the piscine genomes. Although a study has suggested that piscine LXR inhibits intracellular bacterial survival, the functions of piscine LXRα in viral infection are unknown. In this study, we show that overexpression of LXRα from grass carp (Ctenopharyngodon idellus), which is named as gcLXRα, increases host susceptibility to grass carp reovirus (GCRV) infection, whereas gcLXRα knockdown in CIK (C. idellus kidney) cells inhibits GCRV infection. Consistent with these functional studies, gcLXRα knockdown promotes the transcription of antiviral genes involved in the RIG-I-like receptor (RLR) antiviral signaling pathway, including IFN regulatory factor (IRF3) and the type I IFN IFN1. Further results show that gcLXRα knockdown induces the expression of CREB-binding protein (CBP), a transcriptional coactivator. In the knockdown of CBP, the inhibitory effect of gcLXRα knockdown in limiting GCRV infection is completely abolished. gcLXRα also interacts with IRF3 and CBP, which impairs the formation of the IRF3/CBP transcription complex. Moreover, gcLXRα heterodimerizes with RXRg, which cooperatively impair the transcription of the RLR antiviral signaling pathway and promote GCRV infection. Taken together, to our knowledge, our findings provide new insight into the functional correlation between nuclear receptor LXRα and the RLR antiviral signaling pathway, and they demonstrate that gcLXRα can impair the RLR antiviral signaling pathway and the production of type I IFN via forming gcLXRα/RXRg complexes and attenuating IRF3/CBP complexes.

Cisatracurium besylate rescues Mycobacterium Tuberculosis-infected macrophages from necroptosis and enhances the bactericidal effect of isoniazid

ObjectiveTuberculosis is the leading killer among the chronic single-source infectious diseases. Mycobacterium tuberculosis can induce necrotic-dominant multiple modes of cell death in macrophages, which accelerates bacterium dissemination and expands tissue injury in host lungs. Mining drugs to counteract Mycobacterium tuberculosis-induced cell death would be beneficial to tuberculosis patients. MethodsIn this study, the protective drug was screened out from the FDA-approved drug library in Mycobacterium tuberculosis-infected macrophages with CCK-8 assay. The death mode regulated by the drug was identified using transcriptomic sequencing, cytomorphological observation, and in the experimental mouse Mycobacterium tuberculosis-infection model. The functional mechanism was explored using western blot, co-immunoprecipitation, and DARTS assay. The intracellular bacterial survival was detected using colony forming unit assays. ResultsCisatracurium besylate was identified to be highly protective for the viability of macrophages during Mycobacterium tuberculosis infection via inhibiting necroptosis. Cisatracurium besylate prevented RIPK3 to be associated with the executive molecule MLKL for forming the necroptotic complex, resulting in the inhibition of MLKL phosphorylation and pore formation on cell membrane. However, Cisatracurium besylate did not interfere with the association between RIPK3 with its upstream kinase RIPK1 or ZBP1 but regulated RIPK3 autophosphorylation. Moreover, Cisatracurium besylate significantly inhibited the expansion of intracellular Mycobacterium tuberculosis both in vitro and in vivo, which also displayed a strong auxiliary bacteriostatic effect to support the therapeutic efficacy of isoniazid and rifampicin, the first-line anti-tubercular drugs. ConclusionCisatracurium besylate performs anti-Mycobacterium tuberculosis and anti-necroptotic roles, which potentiates its application to be an adjuvant drug for antituberculosis therapy to assist the battle against drug-resistant tuberculosis.

Investigating the lipid-immune signaling axis in modulating the inflammatory response to Salmonella entericaserovar Typhimurium infections

Abstract Salmonella entericaserovar Typhimurium (STM) is an intracellular pathogen that causes gastroenteritis and can disseminate systemically through survival in phagocytes. Eicosanoids-derived bioactive lipids play critical roles in both the induction and resolution of inflammation during bacterial infections or other insults. A subset of these lipids are endogenous ligands for nuclear receptors/transcription factors Peroxisome proliferator-activated receptors (PPAR), which play major roles in fatty acid metabolism and modulation of immune functions (including macrophage MΦ polarization). To elucidate the role of PPARα, we infected streptomycin-pretreated C57BL/6 (wildtype) and Ppara−/−mice with STM (colitis model). We determined that Ppara−/−mice have increased eicosanoid production, decreased inflammatory gene expression and histopathology scores in cecal tissue, and reduced systemic dissemination of bacteria, compared to C57BL/6 mice. These data suggest that the activation of PPARα results in a deleterious immune response favoring STM infection. When polarizing MΦ to various subsets, STM infection enhanced M2b and M2c polarizations (anti-inflammatory and immunoregulatory) in a PPARα-dependent manner. Using liquid chromatography-mass spectrometry (LC/MS), we also determined that STM infection alters the fatty acid profiles in polarized MΦ. Specifically, C57BL/6 M2b-polarized STM-infected MΦ have decreased ceramide levels compared to uninfected control, which would inhibit programmed cell death and enhance bacterial intracellular survival. These findings provide important insights into how the reprogramming of immune metabolism affects host-pathogen interactions during bacterial infections. This work was supported by National Institute of Allergy and Infectious Disease R01 AI168550 (VT)

Porphyromonas gingivalis induction of TLR2 association with Vinculin enables PI3K activation and immune evasion.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that thrives in the inflamed environment of the gingival crevice, and is strongly associated with periodontal disease. The host response to P. gingivalis requires TLR2, however P. gingivalis benefits from TLR2-driven signaling via activation of PI3K. We studied TLR2 protein-protein interactions induced in response to P. gingivalis, and identified an interaction between TLR2 and the cytoskeletal protein vinculin (VCL), confirmed using a split-ubiquitin system. Computational modeling predicted critical TLR2 residues governing the physical association with VCL, and mutagenesis of interface residues W684 and F719, abrogated the TLR2-VCL interaction. In macrophages, VCL knock-down led to increased cytokine production, and enhanced PI3K signaling in response to P. gingivalis infection, effects that correlated with increased intracellular bacterial survival. Mechanistically, VCL suppressed TLR2 activation of PI3K by associating with its substrate PIP2. P. gingivalis induction of TLR2-VCL led to PIP2 release from VCL, enabling PI3K activation via TLR2. These results highlight the complexity of TLR signaling, and the importance of discovering protein-protein interactions that contribute to the outcome of infection.

Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions.

For decades, investigators have studied the interaction of Mycobacterium tuberculosis (Mtb) with macrophages, which serve as a major cellular niche for the bacilli. Because Mtb are prone to aggregation, investigators rely on varied methods to disaggregate the bacteria for these studies. Here, we examined the impact of routinely used preparation methods on bacterial cell envelope integrity, macrophage inflammatory responses, and intracellular Mtb survival. We found that both gentle sonication and filtering damaged the mycobacterial cell envelope and markedly impacted the outcome of infections in mouse bone marrow-derived macrophages. Unexpectedly, sonicated bacilli were hyperinflammatory, eliciting dramatically higher TLR2-dependent gene expression and elevated secretion of IL-1β and TNF-α. Despite evoking enhanced inflammatory responses, sonicated bacilli replicated normally in macrophages. In contrast, Mtb that had been passed through a filter induced little inflammatory response, and they were attenuated in macrophages. Previous work suggests that the mycobacterial cell envelope lipid, phthiocerol dimycocerosate (PDIM), dampens macrophage inflammatory responses to Mtb. However, we found that the impact of PDIM depended on the method used to prepare Mtb. In conclusion, widely used methodologies to disaggregate Mtb may introduce experimental artifacts in Mtb-host interaction studies, including alteration of host inflammatory signaling, intracellular bacterial survival, and interpretation of bacterial mutants.

Neutrophil membrane-coated nanoparticles exhibit increased antimicrobial activities in an anti-microbial resistant K. pneumonia infection model

ObjectiveTo investigate the efficacy and safety of neutrophil membrane-coated nanoparticles mediated KLA peptides (KLAKLAKKLAKLAK) and gentamicin in the targeted therapy of anti-microbial resistant Klebsiella pneumoniae (K. pneumonia) lung infection. MethodsThe characteristics of KLA-neutrophils nanoparticles (NNPs) are identified via dynamic light scattering (DLS), transmission electron microscope (TEM), SDS-PAGE, Western blot, quantitative flow cytometry (QFCM) and confocal microscopy. The safety of KLA-NNPs both in vitro and in vivo is evaluated by hemolysis test, platelet α granule membrane protein concentration, protein adsorption capacity, in vitro macrophage phagocytosis, weight change, liver function indicators, blood biochemical indicators, and pathological changes of vital organs in mice. The efficacy of KLA-NNPs is determined by time-kill assay, fluorescent label test, intracellular bacterial content, caspase-1 activity, survival rate, and HE staining both in vitro and in vivo. ResultsThe prepared KLA-NNPs have a typical “core-shell” structure, uniform nanometer size, and retain the membrane proteins on the neutrophil membrane that achieve functional effects. In vitro safety analysis showed that KLA-NNPs have good blood compatibility and can inhibit macrophage phagocytosis in vitro. KLA-NNPs can effectively release KLA and significantly reduce intracellular bacteria and caspase-1 activity. In vivo safety analysis and efficacy analysis revealed that KLA-NNPs have good biocompatibility and could effectively improve the survival rate of mice. ConclusionThe prepared KLA-NNPs have good nano-medicine chemical and physical properties and safety. It can evade immune system clearance, achieve high-efficiency targeted aggregation and drug delivery to bacterial infection sites, and effectively inhibit the development of pneumonia induced by drug-resistant K. pneumonia.

Potential Pathogenicity of Aeromonas spp. Recovered in River Water, Soil, and Vegetation from a Natural Recreational Area.

The genus Aeromonas is widely distributed in aquatic environments and is recognized as a potential human pathogen. Some Aeromonas species are able to cause a wide spectrum of diseases, mainly gastroenteritis, skin and soft-tissue infections, bacteremia, and sepsis. Currently, untreated river water is used for irrigation and recreational purposes. In this study, the Aeromonas spp. present in a river recreational environment was investigated by quantifying its presence in water, soil, and vegetation using three techniques: qPCR, plate counting in selective ADA medium, and Most Probable Number, in parallel. The presence of clones in the three types of samples was elucidated through genotyping with the ERIC-PCR technique, whereas the identification of the isolated Aeromonas was carried out by sequencing the rpoD gene. Finally, the pathogenic potential of some of the strains was explored by studying the presence and expression of virulence genes characteristic of the genus, their antimicrobial susceptibility profile, as well as the quantification of their cell damage and intracellular survival in an in vitro macrophages infection model. The results showed the presence of Aeromonas in all samples with the three quantification methods, with Aeromonas popoffii being the most prevalent species. The presence of strains with the same genotype (ERIC-PCR) was also confirmed in different samples. Some of the strains showed a high level of cell damage and intracellular bacterial survival, as well as the presence of various virulence factors. Furthermore, these strains showed resistance to some of the antibiotics tested and used therapeutically in both humans and animals. These results indicate that the presence of Aeromonas in this environment may represent a biosanitary risk that could be a public health problem.

Mycobacterium tuberculosis PPE7 Enhances Intracellular Survival of Mycobacterium smegmatis and Manipulates Host Cell Cytokine Secretion Through Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase Signaling.

The PE/PPE family proteins of Mycobacterium tuberculosis have been associated with its virulence and interaction with the host immune system. The highly virulent modern lineage of M. tuberculosis possesses a lineage-specific PPE gene (PPE7), which arises from an ancestral mutation and is rarely studied. Here we examined the role of PPE7 in mycobacterial pathogenicity and survival by expressing M. tuberculosis PPE7 in Mycobacterium smegmatis. We show that, PPE7 activates host inflammation by increasing expression of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6, while suppressing the expression of anti-inflammatory cytokines such as IL-10, possibly through the nuclear factor kappa B, ERK1/2, and p38 mitogen-activated protein kinase pathways. Overexpressing PPE7 in M. smegmatis could enhance bacterial intracellular survival of infected macrophages. Furthermore, higher level of bacterial persistence, higher levels of TNF-α, IL-1β, and IL-6 cytokines, and more injury in the lung, liver, and spleen tissues of infected mice has been discovered. In conclusion, PPE7 could manipulate host immune response and increase bacterial persistence.

Intracellular survival of Streptococcus pneumoniae in human alveolar macrophages is augmented with HIV infection.

People Living with HIV (PLHIV) are at an increased risk of pneumococcal pneumonia than HIV-uninfected adults, but the reasons for this are still not well understood. We investigated whether alveolar macrophages (AM) mediated control of pneumococcal infection is impaired in PLHIV compared to HIV-uninfected adults. We assessed anti-bactericidal activity against Streptococcus pneumoniae of primary human AM obtained from PLHIV and HIV-uninfected adults. We found that pneumococcus survived intracellularly in AMs at least 24 hours post ex vivo infection, and this was more frequent in PLHIV than HIV-uninfected adults. Corroborating these findings, in vivo evidence showed that PLHIV had a higher propensity for harboring S. pneumoniae within their AMs than HIV-uninfected adults. Moreover, bacterial intracellular survival in AMs was associated with extracellular propagation of pneumococcal infection. Our data suggest that failure of AMs to eliminate S. pneumoniae intracellularly could contribute to the increased risk of pneumococcal pneumonia in PLHIV.

A Model of Intracellular Persistence of Pseudomonas aeruginosa in Airway Epithelial Cells

Pseudomonas aeruginosa (P.a.) is a major human pathogen capable of causing chronic infections in hosts with weakened barrier functions and host defenses, most notably airway infections commonly observed in individuals with the genetic disorder cystic fibrosis (CF). While mainly described as an extracellular pathogen, previous in vitro studies have described the molecular events leading to P.a. internalization in diverse epithelial cell types. However, the long-term fate of intracellular P.a. remains largely unknown. Here, we developed a model allowing for a better understanding of long-term (up to 120 h) intracellular bacterial survival in the airway epithelial cell line BEAS-2B. Using a tobramycin protection assay, we characterized the internalization, long-term intracellular survival, and cytotoxicity of the lab strain PAO1, as well as clinical CF isolates, and conducted analyses at the single-cell level using confocal microscopy and flow cytometry techniques. We observed that infection at low multiplicity of infection allows for intracellular survival up to 120 h post-infection without causing significant host cytotoxicity. Finally, infection with clinical isolates revealed significant strain-to-strain heterogeneity in intracellular survival, including a high persistence phenotype associated with bacterial replication within host cells. Future studies using this model will further elucidate the host and bacterial mechanisms that promote P. aeruginosa intracellular persistence in airway epithelial cells, a potentially unrecognized bacterial reservoir during chronic infections.

The group A Streptococcus interleukin-8 protease SpyCEP promotes bacterial intracellular survival by evasion of autophagy.

Autophagy serves an innate immune function in defending the host against invading bacteria, including group A Streptococcus (GAS). Autophagy is regulated by numerous host proteins, including the endogenous negative regulator calpain, a cytosolic protease. Globally disseminated serotype M1T1 GAS strains associated with high invasive disease potential express numerous virulence factors and resist autophagic clearance. Upon in vitro infection of human epithelial cell lines with representative wild-type GAS M1T1 strain 5448 (M1.5448), we observed increased calpain activation linked to a specific GAS virulence factor, the IL-8 protease SpyCEP. Calpain activation inhibited autophagy and decreased capture of cytosolic GAS in autophagosomes. In contrast, the serotype M6 GAS strain JRS4 (M6.JRS4), which is highly susceptible to host autophagy-mediated killing, expresses low levels of SpyCEP and does not activate calpain. Overexpression of SpyCEP in M6.JRS4 stimulated calpain activation, inhibited autophagy and significantly decreased bacterial capture in autophagosomes. These paired loss- and gain-of-function studies reveal a novel role for the bacterial protease SpyCEP in enabling GAS M1 evasion of autophagy and host innate immune clearance.