Infection Of Human Epithelial Cells Research Articles

A synthetic peptide mimic kills Candida albicans and synergistically prevents infection

More than two million people worldwide are affected by life-threatening, invasive fungal infections annually. Candida species are the most common cause of nosocomial, invasive fungal infections and are associated with mortality rates above 40%. Despite the increasing incidence of drug-resistance, the development of novel antifungal formulations has been limited. Here we investigate the antifungal mode of action and therapeutic potential of positively charged, synthetic peptide mimics to combat Candida albicans infections. Our data indicates that these synthetic polymers cause endoplasmic reticulum stress and affect protein glycosylation, a mode of action distinct from currently approved antifungal drugs. The most promising polymer composition damaged the mannan layer of the cell wall, with additional membrane-disrupting activity. The synergistic combination of the polymer with caspofungin prevented infection of human epithelial cells in vitro, improved fungal clearance by human macrophages, and significantly increased host survival in a Galleria mellonella model of systemic candidiasis. Additionally, prolonged exposure of C. albicans to the synergistic combination of polymer and caspofungin did not lead to the evolution of tolerant strains in vitro. Together, this work highlights the enormous potential of these synthetic peptide mimics to be used as novel antifungal formulations as well as adjunctive antifungal therapy.

Tolerance to Haemophilus influenzae infection in human epithelial cells: Insights from a primary cell-based model.

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this, the molecular mechanisms that allow H. influenzae to establish persistent infections of human epithelia are not well understood. Here, we have investigated how H. influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, 'unproductive' inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extracellular and intraepithelial burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations, and appears to involve interruption of NFκB signalling. This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been observed, and we were able to demonstrate that only infections with live, but not heat-killed H. influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1β, IL-36γ and TNFα. Interestingly, NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection, resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H. influenzae in the host, our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation.

Bacterial Pathogen-Mediated Suppression of Host Trafficking to Lysosomes: Fluorescence Microscopy-Based DQ-Red BSA Analysis.

Intracellular bacterial pathogens have evolved to be adept at manipulating host cellular function for the benefit of the pathogen, often by means of secreted virulence factors that target host pathways for modulation. The lysosomal pathway is an essential cellular response pathway to intracellular pathogens and, as such, represents a common target for bacterial-mediated evasion. Here, we describe a method to quantitatively assess bacterial pathogen-mediated suppression of host cell trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This live-cell imaging assay involves the use of a BODIPY TR-X conjugate of BSA (DQ-Red BSA) that traffics to and fluoresces in functional lysosomes. This method can be adapted to study infection with a broad array of pathogens in diverse host cell types. It is capable of being applied to identify secreted virulence factors responsible for a phenotype of interest as well as domains within the bacterial protein that are important for mediating the phenotype. Collectively, these tools can provide invaluable insight into the mechanisms of pathogenesis of a diverse array of pathogenic bacteria, with the potential to uncover virulence factors that may be suitable targets for therapeutic intervention. Key features • Infection-based analysis of bacterial-mediated suppression of host trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of human epithelial cells as a model. • Live microscopy-based analysis allows for the visualization of individually infected host cells and is amenable to phenotype quantification. • Assay can be adapted to a broad array of pathogens and diverse host cell types. • Assay can identify virulence factors mediating a phenotype and protein domains that mediate a phenotype.

Alternative Splicing of RIOK3 Engages the Noncanonical NFκB Pathway during Rift Valley Fever Virus Infection.

Although the noncanonical NFκB pathway was originally identified as a cellular pathway contributing to lymphoid organogenesis, in the past 20 years, its involvement in innate immunity has become more appreciated. In particular, the noncanonical NFκB pathway has been found to be activated and even exploited by some RNA viruses during infection. Intriguingly, activation of this pathway has been shown to have a role in disrupting transcription of type 1 interferon (IFN), suggesting a rationale for why this response could be co-opted by some viruses. Rift Valley fever virus (RVFV) is a trisegmented ambisense RNA virus that poses a considerable threat to domestic livestock and human health. Previously, we showed the atypical kinase RIOK3 is important for mounting an IFN response to RVFV infection of human epithelial cells, and shortly following infection with RVFV (MP12 strain), RIOK3 mRNA is alternatively spliced to its X2 isoform that encodes a truncated RIOK3 protein. Alternative splicing of RIOK3 mRNA has an inhibitory effect on the IFN response but also stimulates an NFκB-mediated inflammatory response. Here, we demonstrate alternative splicing of RIOK3 mRNA is associated with activation of the noncanonical NFκB pathway and suggest this pathway is co-opted by RVFV (MP12) to enhance viral success during infection.

DNA-PKcs restricts Zika virus spreading and is required for effective antiviral response.

Zika virus (ZIKV) is a single-strand RNA mosquito-borne flavivirus with significant public health impact. ZIKV infection induces double-strand DNA breaks (DSBs) in human neural progenitor cells that may contribute to severe neuronal manifestations in newborns. The DNA-PK complex plays a critical role in repairing DSBs and in the innate immune response to infection. It is unknown, however, whether DNA-PK regulates ZIKV infection. Here we investigated the role of DNA-PKcs, the catalytic subunit of DNA-PK, during ZIKV infection. We demonstrate that DNA-PKcs restricts the spread of ZIKV infection in human epithelial cells. Increased ZIKV replication and spread in DNA-PKcs deficient cells is related to a notable decrease in transcription of type I and III interferons as well as IFIT1, IFIT2, and IL6. This was shown to be independent of IRF1, IRF3, or p65, canonical transcription factors necessary for activation of both type I and III interferon promoters. The mechanism of DNA-PKcs to restrict ZIKV infection is independent of DSB. Thus, these data suggest a non-canonical role for DNA-PK during Zika virus infection, acting downstream of IFNs transcription factors for an efficient antiviral immune response.

Human Adenovirus Type 26 Induced IL-6 Gene Expression in an αvβ3 Integrin- and NF-κB-Dependent Manner

The low seroprevalent human adenovirus type 26 (HAdV26)-based vaccine vector was the first adenovirus-based vector to receive marketing authorization from European Commission. HAdV26-based vaccine vectors induce durable humoral and cellular immune responses and, as such, represent a highly valuable tool for fighting infectious diseases. Despite well-described immunogenicity in vivo, the basic biology of HAdV26 still needs some refinement. The aim of this study was to determine the pro-inflammatory cytokine profile of epithelial cells infected with HAdV26 and then investigate the underlying molecular mechanism. The expression of studied genes and proteins was assessed by quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Confocal microscopy was used to visualize HAdV26 cell uptake. We found that HAdV26 infection in human epithelial cells triggers the expression of pro-inflammatory cytokines and chemokines, namely IL-6, IL-8, IL-1β, and TNF-α, with the most pronounced difference shown for IL-6. We investigated the underlying molecular mechanism and observed that HAdV26-induced IL-6 gene expression is αvβ3 integrin dependent and NF-κB mediated. Our findings provide new data regarding pro-inflammatory cytokine and chemokine expression in HAdV26-infected epithelial cells, as well as details concerning HAdV26-induced host signaling pathways. Information obtained within this research increases our current knowledge of HAdV26 basic biology and, as such, can contribute to further development of HAdV26-based vaccine vectors.

Cannabinoids Block Cellular Entry of SARS-CoV-2 and the Emerging Variants.

As a complement to vaccines, small-molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, which cause COVID-19. Affinity selection–mass spectrometry was used for the discovery of botanical ligands to the SARS-CoV-2 spike protein. Cannabinoid acids from hemp (Cannabis sativa) were found to be allosteric as well as orthosteric ligands with micromolar affinity for the spike protein. In follow-up virus neutralization assays, cannabigerolic acid and cannabidiolic acid prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein and prevented entry of live SARS-CoV-2 into cells. Importantly, cannabigerolic acid and cannabidiolic acid were equally effective against the SARS-CoV-2 alpha variant B.1.1.7 and the beta variant B.1.351. Orally bioavailable and with a long history of safe human use, these cannabinoids, isolated or in hemp extracts, have the potential to prevent as well as treat infection by SARS-CoV-2.

ID2 inhibits innate antiviral immunity by blocking TBK1- and IKKε-induced activation of IRF3.

The transcription regulator ID2 plays an essential role in the development and differentiation of immune cells. Here, we report that ID2 also negatively regulates antiviral innate immune responses. During viral infection of human epithelial cells, ID2 bound to TANK-binding kinase 1 (TBK1) and to inhibitor of nuclear factor κB kinase ε (IKKε). These interactions inhibited the recruitment and activation of interferon (IFN) regulatory factor 3 (IRF3) by TBK1 or IKKε, leading to a reduction in the expression of IFN-β1 (IFNB1). IFN-β induced the nuclear export of ID2 to form a negative feedback loop. Knocking out ID2 in human cells enhanced innate immune responses and suppressed infection by different viruses, including SARS-CoV-2. Mice with a myeloid-specific deficiency of ID2 produced more IFN-α in response to viral infection and were more resistant to viral infection than wild-type mice. Our findings not only establish ID2 as a modulator of IRF3 activation induced by TBK1 and/or IKKε but also introduce a mechanism for cross-talk between innate immunity and cell development and differentiation.

The long non-coding RNA landscape of Candida yeast pathogens

Long non-coding RNAs (lncRNAs) constitute a poorly studied class of transcripts with emerging roles in key cellular processes. Despite efforts to characterize lncRNAs across a wide range of species, these molecules remain largely unexplored in most eukaryotic microbes, including yeast pathogens of the Candida clade. Here, we analyze thousands of publicly available sequencing datasets to infer and characterize the lncRNA repertoires of five major Candida pathogens: Candida albicans, Candida tropicalis, Candida parapsilosis, Candida auris and Candida glabrata. Our results indicate that genomes of these species encode hundreds of lncRNAs that show levels of evolutionary constraint intermediate between those of intergenic genomic regions and protein-coding genes. Despite their low sequence conservation across the studied species, some lncRNAs are syntenic and are enriched in shared sequence motifs. We find co-expression of lncRNAs with certain protein-coding transcripts, hinting at potential functional associations. Finally, we identify lncRNAs that are differentially expressed during infection of human epithelial cells for four of the studied species. Our comprehensive bioinformatic analyses of Candida lncRNAs pave the way for future functional characterization of these transcripts.

Shigella ubiquitin ligase IpaH7.8 targets gasdermin D for degradation to prevent pyroptosis and enable infection

The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability.

Immune Transcriptome of Cells Infected with Enterovirus Strains Obtained from Cases of Autoimmune Thyroid Disease

Background: Hashimoto’s thyroiditis and Graves’ disease are autoimmune thyroid disorders (AITD) of unknown origin. Enterovirus (EV) infection of thyroid cells has been implicated as a possible initiator of cell damage and of organ-specific autoimmunity. We asked whether persistent infection of human epithelial cells with EV strains obtained from thyroid tissue of AITD patients could be associated with transcriptional changes capable of fostering immunopathology. Methods: EV isolates obtained from thyroid tissue of AITD cases were used to infect the AV3 epithelial cell line. AV3 cells incubated with a virus-free medium from thyroid tissue of subjects without evidence of thyroid autoimmunity were used as uninfected controls. Transcripts of immune-related genes were compared in infected vs. uninfected cells. Results: The EV genome and antigens were detected only in the cells exposed to AITD-derived virus isolates, not in control cells. Persistent EV infection, while suppressing transcription of several type I IFN and cytokine determinants, was associated with enhanced transcription of NFKB1/RELA, IFNAR1, JAK1/STAT1, i.e., the determinants that play key immunologic roles. Infection also led to upregulation of the CCL2 chemokine and the IL-18 pro-inflammatory interleukin. Conclusion: As in the case of EV strains obtained from autoimmune diabetes, results show that the EV strains that are present in the thyroid of AITD cases do repress IFN and cytokine pathways. JAK1/STAT1 upregulation supports activation of TLR pathways and aberrant T cell signaling. In the early phases of AITD, our results highlight the potential benefit of interventions aimed at blocking the viral infection and easing the inflammatory response.

Facile accelerated specific therapeutic (FAST) platform develops antisense\xa0therapies to counter multidrug-resistant bacteria

Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpetuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs. As a proof of concept, these PNAs were tested against five MDR clinical isolates: carbapenem-resistant Escherichia coli, extended-spectrum beta-lactamase Klebsiella pneumoniae, New Delhi Metallo-beta-lactamase-1 carrying Klebsiella pneumoniae, and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of treatments leading to greater than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, the FAST platform offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells by repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellular Salmonella infection in human epithelial cells.

Characterization of Listeria monocytogenes isolates from lactating dairy cows in a Wisconsin farm: Antibiotic resistance, mammalian cell infection, and effects on the fecal microbiota

Listeria monocytogenes is an invasive foodborne pathogen that is ubiquitously present in the dairy farm environment. Although cattle are a reservoir of L. monocytogenes, most adult animals do not exhibit clinical symptoms, suggesting a homeostasis between this pathogen and the bovine gastrointestinal ecosystem. Nevertheless, substantial prevalence of L. monocytogenes fecal shedding by dairy cattle has been reported in many studies, posing threats of transmission within the herd and contamination of the human food supply. Accordingly, understanding the L. monocytogenes ecology within the bovine gastrointestinal tract is important to prevent clinical illness in the animal host, reduce transmission, and guide intervention strategies. In this study, we conducted a longitudinal sampling of fecal samples from 20 lactating dairy cows in one Wisconsin farm over a 29-d period and found a strikingly high incidence of L. monocytogenes shedding, in 90% of sampled animals. The L. monocytogenes isolates were genetically diverse, representing all common serotypes previously identified from cattle. Additionally, most tested isolates were resistant to ampicillin, and a few were also resistant to gentamicin or trimethoprim/sulfamethoxazole. Most isolates effectively infected human epithelial cells (Caco-2) and murine fibroblasts (L2), suggesting that they are all capable of causing systemic infection if the intestinal barrier is breached. Finally, we investigated the effects of L. monocytogenes colonization on the gastrointestinal tract microbiota by analyzing the fecal bacterial communities of some shedding and nonshedding cows. Whereas L. monocytogenes did not affect the α and β diversity of tested animals, a subset of shedding cows exhibited different abundances of certain operational taxonomic units within the Bacteroidetes and Firmicutes phyla compared with nonshedding cows. Overall, our findings highlight the threat of antibiotic resistance among some L. monocytogenes isolates, emphasize the need for a strain-specific approach in listeriosis treatment, and suggest the potential negative influence of subclinical L. monocytogenes carriage on animal gut health.

Baculovirus Transduction in Mammalian Cells Is Affected by the Production of Type I and III Interferons, Which Is Mediated Mainly by the cGAS-STING Pathway.

The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of HEK293 and HEK293 T human epithelial cells, deficient in cGAS and in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted in the more relevant production of beta interferon (IFN-β) and IFN-λ1 in response to baculovirus infection. In human epithelial cells, IFN-λ1 production was also induced in a cGAS-independent and DNA-protein kinase (DNA-PK)-dependent manner. Finally, we demonstrated that these cellular responses toward baculovirus infection affect the efficiency of transduction of baculovirus vectors.IMPORTANCE Baculoviruses are nonpathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and type III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.

Context-Dependent Action of Scc4 Reinforces Control of the Type III Secretion System.

Chlamydia trachomatis Scc4 (formerly CT663) engages the transcription machinery and the pathogenic type III secretion system (T3SS). Both machines are required for Chlamydia infection. These requirements and the limited ability for genetic manipulation in Chlamydia have hampered dissection of Scc4's contributions. Here, by developing bacterial systems that permit the controlled expression and stable maintenance of Scc4, we assess Scc4's effects on chlamydial growth phenotype, secretion, and the patterns of T3SS gene expression. Expressing Scc4 in Escherichia coli lacking a T3SS injectisome causes a growth defect. This deficiency is rescued by overexpressing the β-subunit of RNA polymerase (RNAP) or by exploiting sigma 70 (σ70) (homologous to chlamydial σ66) mutants that strengthen the interaction between σ70 region 4 and the β-flap, confirming Scc4's distinction as a module of RNAP holoenzyme capable of modulating transcription. Yersinia pestis expressing Scc4 sustains a functional T3SS, through which CopN secretion is boosted by cooption of Scc4 and Scc1. Finally, conditional expression of Scc4 in C. trachomatis results in fast expansion of the Chlamydia-containing vacuole and accelerated chlamydial development, coupled to selective up- or downregulation of gene expression from different T3SS genes. This work reveals, for the first time, the context-dependent action of Scc4 linking it to diverse protein networks in bacteria. It establishes that Scc4, when overexpressed, exerts incredible effects on chlamydial development by reinforcing control of the T3SS.IMPORTANCE The T3SS is a key virulence factor required for C. trachomatis infection. The control of the T3SS has not been well studied in this obligate intracellular pathogen. Here, we show that Scc4 plays a major role for precise control of the pathogenic T3SS at the levels of gene expression and effector secretion through genetically separable protein networks, allowing a fast adaptive mode of C. trachomatis development during infection in human epithelial cells.

Analysis of Virus and Host Proteomes During Productive HSV-1 and VZV Infection in Human Epithelial Cells.

Herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) are two closely related human alphaherpesviruses that persistently infect most adults worldwide and cause a variety of clinically important diseases. Herpesviruses are extremely well adapted to their hosts and interact broadly with cellular proteins to regulate virus replication and spread. However, it is incompletely understood how HSV-1 and VZV interact with the host proteome during productive infection. This study determined the temporal changes in virus and host protein expression during productive HSV-1 and VZV infection in the same cell type. Results demonstrated the temporally coordinated expression of HSV-1 and VZV proteins in infected cells. Analysis of the host proteomes showed that both viruses affected extracellular matrix composition, transcription, RNA processing and cell division. Moreover, the prominent role of epidermal growth factor receptor (EGFR) signaling during productive HSV-1 and VZV infection was identified. Stimulation and inhibition of EGFR leads to increased and decreased virus replication, respectively. Collectively, the comparative temporal analysis of viral and host proteomes in productively HSV-1 and VZV-infected cells provides a valuable resource for future studies aimed to identify target(s) for antiviral therapy development.

Chaperone activity of serine protease HtrA of Helicobacter pylori as a crucial survival factor under stress conditions

BackgroundSerine protease HtrA exhibits both proteolytic and chaperone activities, which are involved in cellular protein quality control. Moreover, HtrA is an important virulence factor in many pathogens including Helicobacter pylori, for which the crucial stage of infection is the cleavage of E-cadherin and other cell-to-cell junction proteins.MethodsThe in vitro study of H. pylori HtrA (HtrAHp) chaperone activity was carried out using light scattering assays and investigation of lysozyme protein aggregates. We produced H. pylori ∆htrA deletion and HtrAHp point mutants without proteolytic activity in strain N6 and investigated the survival of the bacteria under thermal, osmotic, acidic and general stress conditions as well as the presence of puromycin or metronidazole using serial dilution tests and disk diffusion method. The levels of cellular and secreted proteins were examined using biochemical fraction and Western blotting. We also studied the proteolytic activity of secreted HtrAHp using zymography and the enzymatic digestion of β-casein. Finally, the consequences of E-cadherin cleavage were determined by immunofluorescence microscopy.ResultsWe demonstrate that HtrAHp displays chaperone activity that inhibits the aggregation of lysozyme and is stable under various pH and temperature conditions. Next, we could show that N6 expressing only HtrA chaperone activity grow well under thermal, pH and osmotic stress conditions, and in the presence of puromycin or metronidazole. In contrast, in the absence of the entire htrA gene the bacterium was more sensitive to a number of stresses. Analysing the level of cellular and secreted proteins, we noted that H. pylori lacking the proteolytic activity of HtrA display reduced levels of secreted HtrA. Moreover, we compared the amounts of secreted HtrA from several clinical H. pylori strains and digestion of β-casein. We also demonstrated a significant effect of the HtrAHp variants during infection of human epithelial cells and for E-cadherin cleavage.ConclusionHere we identified the chaperone activity of the HtrAHp protein and have proven that this activity is important and sufficient for the survival of H. pylori under multiple stress conditions. We also pinpointed the importance of HtrAHp chaperone activity for E- cadherin degradation and therefore for the virulence of this eminent pathogen.

Frequency and expression of genes involved in adhesion and biofilm formation in Staphylococcus aureus strains isolated from periodontal lesions.

The aim of this study was to characterize the Staphylococcus aureus strains isolated from periodontal lesions of patients, to determine the expression of genes involved in cell adhesion upon their infection of human epithelial cells using an invitro model, its biofilm formation, and its resistance to antibiotics. S. aureus was analysed by PCR, Kirby-Bauer, and pulsed-field gel electrophoresis (PFGE), measuring gene expression by real-time PCR after infection of human cells invitro. S. aureus was identified in 18.6% (50/268) of the samples. All strains (n=50) possessed the virulence genes spa (Staphylococcal protein A), coa (coagulase), and icaAB (intercellular adhesin); 96% (n=48) possessed clfB (clumping factor B), and 88% (n=44) possessed ebps (elastin-binding protein) and sdrD (serine aspartate repeat protein D). All strains were resistant to methicillin, ampicillin, dicloxacillin, cefotaxime, and penicillin, and were multidrug resistant to 6-12 antibiotics. PFGE analysis showed 37 different pulsed-field types and most strains (60.4%) had a unique pulsed-field type. Twenty-four distinct combinations of virulence genes and antibiotic-resistant phenotypes were identified. Although S. aureus has been considered a transient member of the oral microbiota, our results indicate a high-level expression of virulence genes and multidrug resistance in the strains isolated from periodontal lesions. These strains might complicate the successful treatment of the disease.

Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Analysis of Staphylococcus aureus proteins secreted inside infected human epithelial cells

Staphylococcus aureus, an opportunistic pathogen is able to invade into and persist inside non-professional phagocytic cells. To do so, this bacterium possesses a wide range of secreted virulence factors which enable attachment to the host as well as intracellular survival. Hence, a monitoring of virulence factors specifically produced upon internalization might reveal targets for prevention or therapy of S. aureus infections. However, previous proteome approaches enriching S. aureus from lysed host cells after infection did not cover secreted virulence factors.Therefore, we used density gradient centrifugation and mass spectrometry to identify S. aureus HG001 proteins which were secreted into compartments of infected human bronchial epithelial S9 cells. Because shotgun mass spectrometry revealed only few bacterial proteins amongst 1905 host proteins, we used highly sensitive and selective single reaction monitoring mass spectrometry as an alternative approach and quantified 37 bacterial proteins within the S. aureus containing host cell compartment 2.5 h and 6.5 h post infection. Among them were secreted bacterial virulence factors like lipases, pore forming toxins, and secreted adhesins which are usually hard to detect from infected sample material by proteomics approaches due to their low abundance. S. aureus adapted its proteome to improve its response to oxidative and cell wall stress occurring inside the host, but also, increased the amounts of some adhesins and pore-forming toxins, required for attachment and host cell lysis.