Adhesion of Klebsiella oxytoca to bladder or lung epithelial cells is promoted by the presence of other opportunistic pathogens.

The intestinal and respiratory tracts of healthy individuals serve as habitats for a diverse array of microorganisms, among which Klebsiella oxytoca holds significance as a causative agent in numerous community- and hospital-acquired infections, often manifesting in polymicrobial contexts. In specific circumstances, K. oxytoca, alongside other constituents of the gut microbiota, undergoes translocation to distinct physiological niches. In these new environments, it engages in close interactions with other microbial community members. As this interaction may progress to co-infection where the virulence of involved pathogens may be promoted and enhance disease severity, we investigated how K. oxytoca affects the adhesion of commonly co-isolated bacteria and vice versa during co-incubation of different biotic and abiotic surfaces. Co-incubation was beneficial for the adhesion of at least one of the two co-cultured strains. K. oxytoca enhanced the adhesion of other enterobacteria strains to polystyrene and adhered more efficiently to bladder or lung epithelial cell lines in the presence of most enterobacteria strains and S. aureus. This effect was accompanied by bacterial coaggregation mediated by carbohydrate-protein interactions occurring between bacteria. These interactions occur only in sessile, but not planktonic populations, and depend on the features of the surface. The data are of particular importance for the risk assessment of the urinary and respiratory tract infections caused by K. oxytoca, including those device-associated. In this paper, we present the first report on K. oxytoca ability to acquire increased adhesive capacities on epithelial cells through interactions with common causal agents of urinary and respiratory tract infections.

Bacterial-derived sialidases inhibit porcine rotavirus OSU replication by interfering with the early steps of infection.

Rotavirus infections in suckling and weaning piglets cause severe dehydration and death, resulting in significant economic losses in the pig breeding industry. With the continuous emergence of porcine rotavirus (PoRV) variants and poor vaccine cross-protection among various genotypes, there is an urgent need to develop alternative strategies such as seeking effective antiviral products from nature, microbial metabolites and virus-host protein interaction. Sialidases play a crucial role in various physiopathological processes and offer a promising target for developing antivirus drugs. However, the effect of bacterial-derived sialidases on the infection of PoRVs remains largely unknown. Herein, we investigated the impact of bacterial-derived sialidases (sialidase Cp and Vc) on PoRV strain OSU(Group A) infection, using differentiated epithelial monkey kidney cells (MA104) as a model. Our results indicated that the pretreatment of MA104 with exogenous sialidases effectively suppressed PoRV OSU in a concentration-dependent manner. Notably, even at a concentration of 0.01 µU/mL, sialidases significantly inhibited the virus (MOI = 0.01). Meanwhile, we found that sialidase Vc pretreatment sharply reduced the binding rate of PoRV OSU. Last, we demonstrated that PoRV OSU might recognize α-2,3-linked sialic acid as the primary attachment factor in MA104. Our findings provide new insights into the underlying mechanism of PoRV OSU infections, shedding lights on the development of alternative antivirus approaches based on bacteria-virus interaction.

The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion.

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32-0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6.

Gastrointestinal signals in supplemented media reveal a role in adherence for the Shigella flexneri sap autotransporter gene.

Shigella flexneri causes severe diarrheal disease worldwide. While many aspects of pathogenesis have been elucidated, significant knowledge gaps remain regarding the role of putative chromosomally-encoded virulence genes. The uncharacterized sap gene encoded on the chromosome has significant nucleotide sequence identity to the fluffy (flu) antigen 43 autotransporter gene in pathogenic Escherichia coli. Here, we constructed a Δsap mutant in S. flexneri strain 2457T and examined the effects of this mutation on bacterial cell aggregation, biofilm formation, and adherence to colonic epithelial cells. Analyses included the use of growth media supplemented with glucose and bile salts to replicate small intestinal signals encountered by S. flexneri. Deletion of the sap gene in 2457T affected epithelial cell adherence, resulted in quicker bacterial cell aggregation, but did not affect biofilm formation. This work highlights a functional role for the sap gene in S. flexneri pathogenesis and further demonstrates the importance of using relevant and appropriate gastrointestinal signals to characterize virulence genes of enteropathogenic bacteria.

Genes and pathways revealed by whole transcriptome analysis of milk derived bovine mammary epithelial cells after Escherichia coli challenge.

Mastitis, inflammation of the mammary gland, is the costliest disease in dairy cattle and a major animal welfare concern. Mastitis is usually caused by bacteria, of which staphylococci, streptococci and Escherichia coli are most frequently isolated from bovine mastitis. Bacteria activate the mammary immune system in variable ways, thereby influencing the severity of the disease. Escherichia coli is a common cause of mastitis in cattle causing both subclinical and clinical mastitis. Understanding of the molecular mechanisms that activate and regulate the host response would be central to effective prevention of mastitis and breeding of cows more resistant to mastitis. We used primary bovine mammary epithelial cell cultures extracted noninvasively from bovine milk samples to monitor the cellular responses to Escherichia coli challenge. Differences in gene expression between control and challenged cells were studied by total RNA-sequencing at two time points post-challenge. In total, 150 and 440 (Padj < 0.05) differentially expressed genes were identified at 3 h and 24 h post-challenge, respectively. The differentially expressed genes were mostly upregulated at 3 h (141/150) and 24 h (424/440) post-challenge. Our results are in line with known effects of E. coli infection, with a strong early inflammatory response mediated by pathogen receptor families. Among the most significantly enriched early KEGG pathways were the TNF signalling pathway, the cytokine-cytokine receptor interaction, and the NF-kappa B signalling pathway. At 24 h post-challenge, most significantly enriched were the Influenza A, the NOD-like receptor signalling, and the IL-17 signaling pathway.

Aerobic glycolysis of bronchial epithelial cells rewires Mycoplasma pneumoniae pneumonia and promotes bacterial elimination.

The immune response to Mycoplasma pneumoniae infection plays a key role in clinical symptoms. Previous investigations focused on the pro-inflammatory effects of leukocytes and the pivotal role of epithelial cell metabolic status in finely modulating the inflammatory response have been neglected. Herein, we examined how glycolysis in airway epithelial cells is affected by M. pneumoniae infection in an in vitro model. Additionally, we investigated the contribution of ATP to pulmonary inflammation. Metabolic analysis revealed a marked metabolic shift in bronchial epithelial cells during M. pneumoniae infection, characterized by increased glucose uptake, enhanced aerobic glycolysis, and augmented ATP synthesis. Notably, these metabolic alterations are orchestrated by adaptor proteins, MyD88 and TRAM. The resulting synthesized ATP is released into the extracellular milieu via vesicular exocytosis and pannexin protein channels, leading to a substantial increase in extracellular ATP levels. The conditioned medium supernatant from M. pneumoniae-infected epithelial cells enhances the secretion of both interleukin (IL)-1ß and IL-18 by peripheral blood mononuclear cells, partially mediated by the P2X7 purine receptor (P2X7R). In vivo experiments confirm that addition of a conditioned medium exacerbates pulmonary inflammation, which can be attenuated by pre-treatment with a P2X7R inhibitor. Collectively, these findings highlight the significance of airway epithelial aerobic glycolysis in enhancing the pulmonary inflammatory response and aiding pathogen clearance.

Interleukin-22 enhanced the mucosal barrier and inhibited the invasion of Salmonella enterica in human-induced pluripotent stem cell-derived small intestinal epithelial cells.

Human-induced pluripotent stem cell-derived small intestinal epithelial cell (hiPSC-SIEC) monolayers are useful in vitro models for evaluating the gut mucosal barrier; however, their reactivity to cytokines, which are closely related to the regulation of mucosal barrier function, remains unclear. Interleukin (IL)-22 is a cytokine that contributes to regulate the mucosal barrier in the intestinal epithelia. Using microarray and gene set enrichment analysis, we found that hiPSC-SIEC monolayers activate the immune response and enhance the mucosal barrier in response to IL-22. Moreover, hiPSC-SIEC monolayers induced the gene expression of antimicrobials, including the regenerating islet-derived protein 3 family. Furthermore, IL-22 stimulation upregulated Mucin 2 secretion and gene expression of an enzyme that modifies sugar chains, suggesting alteration of the state of the mucus layer of hiPSC-SIEC monolayers. To evaluate its physiological significance, we measured the protective activity against Salmonella enterica subsp. enterica infection in hiPSC-SIEC monolayers and found that prestimulation with IL-22 reduced the number of viable intracellular bacteria. Collectively, these results suggest that hiPSC-SIEC monolayers enhance the mucosal barrier and inhibit infection by pathogenic bacteria in response to IL-22, as previously reported. These results can contribute to the further application of hiPSC-SIECs in evaluating mucosal barriers.

GH136-encoding gene (perB) is involved in gut colonization and persistence by Bifidobacterium bifidum PRL2010.

Bifidobacteria are commensal microorganisms that typically inhabit the mammalian gut, including that of humans. As they may be vertically transmitted, they commonly colonize the human intestine from the very first day following birth and may persist until adulthood and old age, although generally at a reduced relative abundance and prevalence compared to infancy. The ability of bifidobacteria to persist in the human intestinal environment has been attributed to genes involved in adhesion to epithelial cells and the encoding of complex carbohydrate-degrading enzymes. Recently, a putative mucin-degrading glycosyl hydrolase belonging to the GH136 family and encoded by the perB gene has been implicated in gut persistence of certain bifidobacterial strains. In the current study, to better characterize the function of this gene, a comparative genomic analysis was performed, revealing the presence of perB homologues in just eight bifidobacterial species known to colonize the human gut, including Bifidobacterium bifidum and Bifidobacterium longum subsp. longum strains, or in non-human primates. Mucin-mediated growth and adhesion to human intestinal cells, in addition to a rodent model colonization assay, were performed using B. bifidum PRL2010 as a perB prototype and its isogenic perB-insertion mutant. These results demonstrate that perB inactivation reduces the ability of B. bifidum PRL2010 to grow on and adhere to mucin, as well as to persist in the rodent gut niche. These results corroborate the notion that the perB gene is one of the genetic determinants involved in the persistence of B. bifidum PRL2010 in the human gut.

Temperature influences commensal-pathogen dynamics in a nasal epithelial cell co-culture model.

Chronic rhinosinusitis (CRS) is an inflammatory disease of the paranasal sinuses, and microbial dysbiosis associated with CRS is thought to be a key driver of host inflammation that contributes to disease progression. Staphylococcus aureus is a common upper respiratory tract (URT) pathobiont associated with higher carriage rates in CRS populations, where S. aureus-secreted toxins can be identified in CRS tissues. Although many genera of bacteria colonize the URT, few account for the majority of sequencing reads. These include S. aureus and several species belonging to the genus Corynebacterium, including Corynebacterium propinquum and Corynebacterium pseudodiphtheriticum, which are observed at high relative abundance in the healthy URT. Studies have examined bacterial interactions between major microbionts of the URT and S. aureus, but few have done so in the context of a healthy versus diseased URT environment. Here, we examine the role of temperature in commensal, pathogen, and epithelial dynamics using an air-liquid interface cell culture model mimicking the nasal epithelial environment. Healthy URT temperatures change from the nares to the nasopharynx and are increased during disease. Temperatures representative of the healthy URT increase persistence and aggregate formation of commensal C. propinquum and C. pseudodiphtheriticum, reduce S. aureus growth, and lower epithelial cytotoxicity compared to higher temperatures correlating with the diseased CRS sinus. Dual-species colonization revealed species-specific interactions between Corynebacterium species and S. aureus dependent on temperature. Our findings suggest URT mucosal temperature plays a significant role in mediating polymicrobial and host-bacterial interactions that may exacerbate microbial dysbiosis in chronic URT diseases.IMPORTANCEChronic rhinosinusitis is a complex inflammatory disease with a significant healthcare burden. Although presence of S. aureus and microbial dysbiosis are considered mediators of inflammation in CRS, no studies have examined the influence of temperature on S. aureus interactions with the nasal epithelium and the dominant genus of the healthy URT, Corynebacterium. Interactions between Corynebacterium species and S. aureus have been documented in several studies, but none to date have examined how environmental changes in the URT may alter their interactions with the epithelium or each other. This study utilizes a polarized epithelial cell culture model at air-liquid interface to study the colonization and spatial dynamics of S. aureus and clinical isolates of Corynebacterium from people with CRS to characterize the role temperature has in single- and dual-species dynamics on the nasal epithelium.

TRAF3 gene regulates macrophage migration and activation by lung epithelial cells infected with Aspergillus fumigatus.

IMPORTANCE: Aspergillus fumigatus can infect immunocompromised individuals and cause chronic and fatal invasive fungal infections. A better understanding of the molecular mechanisms of A. fumigatus-host interactions may provide new references for disease treatment. In this study, we demonstrated that the TRAF3 gene plays an important role in the early infection of A. fumigatus by regulating the resistance of lung epithelial cells to A. fumigatus. Macrophages are the most abundant innate immune cells in the alveoli; however, few studies have reported on the interactions between lung epithelial cells and macrophages in response to A. fumigatus invasion. In our study, it was demonstrated that the TRAF3 gene reduces migration to macrophages and cytokine production by negatively regulating lung epithelial cell adhesion and internalization of A. fumigatus spores. Together, our results provide new insights into lung epithelial cell-macrophage interactions during A. fumigatus infection.

PFKFB3-Meditated Glycolysis via the Reactive Oxygen Species-Hypoxic Inducible Factor 1α Axis Contributes to Inflammation and Proliferation of Staphylococcus aureus in Epithelial Cells.

Mastitis caused by antibiotic-resistant strains of Staphylococcus aureus is a significant concern in the livestock industry due to the economic losses it incurs. Regulating immunometabolism has emerged as a promising approach for preventing bacterial inflammation. To investigate the possibility of alleviating inflammation caused by S aureus infection by regulating host glycolysis, we subjected the murine mammary epithelial cell line (EpH4-Ev) to S aureus challenge. Our study revealed that S aureus can colonize EpH4-Ev cells and promote inflammation through hypoxic inducible factor 1α (HIF1α)-driven glycolysis. Notably, the activation of HIF1α was found to be dependent on the production of reactive oxygen species (ROS). By inhibiting PFKFB3, a key regulator in the host glycolytic pathway, we successfully modulated HIF1α-triggered metabolic reprogramming by reducing ROS production in S aureus-induced mastitis. Our findings suggest that there is a high potential for the development of novel anti-inflammatory therapies that safely inhibit the glycolytic rate-limiting enzyme PFKFB3.

Synergistic Inhibitory Effect of Lactobacillus Cell Lysates and Butyrate on Poly I:C-Induced IL-8 Production in Human Intestinal Epithelial Cells.

Postbiotics include cell lysates (CLs), enzymes, cell wall fragments, and heat-killed bacteria derived from probiotics. Although postbiotics are increasingly being considered for their potential health-promoting properties, the effects of postbiotics on virus-mediated inflammatory responses in the intestine have not been elucidated. Hence, the present study aimed to examine whether CLs of Lactipantibacillus plantarum (LP CL) and Lacticaseibacillus rhamnosus GG (LR CL) could inhibit virus-mediated inflammatory responses in the human intestinal epithelial cell line HT-29 in vitro. Pretreatment with LP CL and LR CL significantly inhibited interleukin (IL)-8 production, which was induced by poly I:C, a synthetic analog of double-stranded RNA (dsRNA) viruses, at the mRNA and protein levels in HT-29 cells. However, peptidoglycans and heat-killed L. plantarum and L. rhamnosus GG did not effectively inhibit IL-8 production. LP CL and LR CL attenuated the poly I:C-induced phosphorylation of ERK and JNK and the activation of NF-κB, suggesting that these CLs could inhibit poly I:C-induced IL-8 production by regulating intracellular signaling pathways in HT-29 cells. Furthermore, among the short-chain fatty acids, butyrate enhanced the inhibitory effect of CLs on poly I:C-induced IL-8 production at the mRNA and protein levels in HT-29 cells, while acetate and propionate did not. Taken together, these results suggest that both LP CL and LR CL could act as potent effector molecules that can inhibit virus-mediated inflammatory responses and confer synergistic inhibitory effects with butyrate in human intestinal epithelial cells.

Bordetella pertussis targets the basolateral membrane of polarized respiratory epithelial cells, gets internalized, and survives in intracellular locations.

The airway epithelial barrier is a continuous highly organized cell layer that separates the exterior from the underlying mucosal tissue, preventing pathogen invasion. Several respiratory pathogens have evolved mechanisms to compromise this barrier, invade and even reside alive within the epithelium. Bordetella pertussis is a persistent pathogen that infects the human airway epithelium, causing whooping cough. Previous studies have shown that B. pertussis survives inside phagocytic and nonphagocytic cells, suggesting that there might be an intracellular stage involved in the bacterial infectious process and/or in the pathogen persistence inside the host. In this study we found evidence that B. pertussis is able to survive inside respiratory epithelial cells. According to our results, this pathogen preferentially attaches near or on top of the tight junctions in polarized human bronchial epithelial cells and disrupts these structures in an adenylate cyclase-dependent manner, exposing their basolateral membrane. We further found that the bacterial internalization is significantly higher in cells exposing this membrane compared with cells only exposing the apical membrane. Once internalized, B. pertussis mainly remains in nondegradative phagosomes with access to nutrients. Taken together, these results point at the respiratory epithelial cells as a potential niche of persistence.

Bacterial outer membrane vesicles provide an alternative pathway for trafficking of Escherichia coli O157 type III secreted effectors to epithelial cells.

IMPORTANCE: Bacteria can package protein cargo into nanosized membrane blebs that are shed from the bacterial membrane and released into the environment. Here, we report that a type of pathogenic bacteria called enterohemorrhagic Escherichia coli O157 (EHEC) uses their membrane blebs (outer membrane vesicles) to package components of their type 3 secretion system and send them into host cells, where they can manipulate host signaling pathways including those involved in infection response, such as immunity. Usually, EHEC use a needle-like apparatus to inject these components into host cells, but packaging them into membrane blebs that get taken up by host cells is another way of delivery that can bypass the need for a functioning injection system.

Upregulation of occludin by cytolethal distending toxin facilitates Glaesserella parasuis adhesion to respiratory tract cells.

Virulent Glaesserella parasuis may engender systemic infection characterized by fibrinous polyserositis and pneumonia. G. parasuis causes systemic disease through upper respiratory tract infection, but the mechanism has not been fully characterized. Tight junction (TJ) proteins maintain the integrity and impermeability of the epithelial barriers. In this work, we applied the recombinant cytolethal distending toxin (CDT) holotoxin and cdt-deficient mutants to assess whether CDT interacted with TJ proteins of airway tract cells. Our results indicated that CDT induced the TJ occludin (OCLN) expression in newborn pig tracheal epithelial cells within the first 3 hours of bacterial infection, followed by a significant decrease. Overexpression of OCLN in target cells made them more susceptible to G. parasuis adhesion, whereas ablation of OCLN expression by CRISPR/Cas 9 gene editing technology in target cells decreased their susceptibility to bacterial adhesion. In addition, CDT treatment could upregulate the OCLN levels in the lung tissue of C57/BL6 mice. In summary, highly virulent G. parasuis strain SC1401 stimulated the tight junction expression, resulting in higher bacterial adhesion to respiratory tract cells, and this process is closely related to CDT. Our results may provide novel insights into G. parasuis infection and CDT-mediated pathogenesis.

Molecular mechanism of Enteroaggregative Escherichia coli induced apoptosis in cultured human intestinal epithelial cells.

Background. Enteroaggregative Escherichia coli (EAEC) is an evolving etiological agent of acute and persistent diarrhoea worldwide. The previous study from our laboratory has reported the apoptosis-inducing activity of EAEC in human small intestinal and colonic epithelial cell lines. In the present investigation, we have explored the underlying mechanism of EAEC-induced apoptosis in human intestinal epithelial cell lines.Methods. INT-407 and HCT-15 cells were infected with EAEC-T8 and EAEC-pT8 (plasmid cured strain of EAEC-T8) separately. Cells cultured in the absence of bacteria served as a negative control in all the experiments. For the subsequent experiments, the molecular mechanism(s) of epithelial cell aposptosis was measured in EAEC infecting both the cell lines by flow cytometry, real-time PCR and Western blotting.Results and conclusions. EAEC was found to activate the intrinsic/mitochondrial apoptotic pathway in both the cell lines through upregulation of pro-apoptotic Bax and Bak, un-alteration/reduction in the level of anti-apoptotic Bcl-2 and Bcl-XL, decrease in mitochondrial transmembrane potential, accumulation of cytosolic cytochrome c leading to activation of procaspase-9 and procaspase-3, which ultimately resulted in DNA fragmentation and apoptosis. Further, an increased expression of Fas, activation of procaspase-8 and upregulation of pro-apoptotic Bid in the EAEC-infected cells indicated the involvement of extrinsic apoptotic pathway too in this process. Our finding has undoubtedly led to an increased understanding of EAEC pathogenesis, which may be helpful to develop an improved strategy to combat the infection.

Identification of differences in gene expression implicated in the Adherent-Invasive Escherichia coli phenotype during in vitro infection of intestinal epithelial cells.

Introduction: Adherent-invasive Escherichia coli (AIEC) is strongly associated with the pathogenesis of Crohn's disease (CD). However, no molecular markers currently exist for AIEC identification. This study aimed to identify differentially expressed genes (DEGs) between AIEC and non-AIEC strains that may contribute to AIEC pathogenicity and to evaluate their utility as molecular markers. Methods: Comparative transcriptomics was performed on two closely related AIEC/non-AIEC strain pairs during Intestine-407 cell infection. DEGs were quantified by RT-qPCR in the same RNA extracts, as well as in 14 AIEC and 23 non-AIEC strains to validate the results across a diverse strain collection. Binary logistical regression was performed to identify DEGs whose quantification could be used as AIEC biomarkers. Results: Comparative transcriptomics revealed 67 differences in expression between the two phenotypes in the strain pairs, 50 of which (81.97%) were corroborated by RT-qPCR. When explored in the whole strain collection, 29 DEGs were differentially expressed between AIEC and non-AIEC phenotypes (p-value < 0.042), and 42 genes between the supernatant fraction of infected cell cultures and the cellular fraction containing adhered and intracellular bacteria (p-value < 0.049). Notably, six DEGs detected in the strain collection were implicated in arginine biosynthesis and five in colanic acid synthesis. Furthermore, two biomarkers based on wzb and cueR gene expression were proposed with an accuracy of ≥ 85% in our strain collection. Discussion: This is the first transcriptomic study conducted using AIEC-infected cell cultures. We have identified several genes that may be involved in AIEC pathogenicity, two of which are putative biomarkers for identification.

Intracellular replication of Pseudomonas aeruginosa in epithelial cells requires suppression of the caspase-4 inflammasome.

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

Use of a human small airway epithelial cell line to study the interactions of Aspergillus fumigatus with pulmonary epithelial cells.

During the initiation of invasive aspergillosis, inhaled Aspergillus fumigatus conidia are deposited on the epithelial cells lining the bronchi, terminal bronchioles, and alveoli. While the interactions of A. fumigatus with bronchial and type II alveolar cell lines have been investigated in vitro, little is known about the interactions of this fungus with terminal bronchiolar epithelial cells. Using the HSAEC1-KT human small airway epithelial (HSAE) cell line, we developed an in vitro model to study the interaction of two strains of A. fumigatus with these cells. We then compared the interactions of A. fumigatus with the A549 type II alveolar epithelial cell line and the HSAE cell line. We found that A. fumigatus conidia were poorly endocytosed by A549 cells, but avidly endocytosed by HSAE cells. A. fumigatus germlings invaded both cell types by induced endocytosis, but not by active penetration. A549 cell endocytosis of A. fumigatus was independent of fungal viability, more dependent on host microfilaments than microtubules, and induced by A. fumigatus CalA interacting with host cell integrin α5ß1. By contrast, HSAE cell endocytosis required fungal viability, was more dependent on microtubules than microfilaments, and did not require CalA or integrin α5ß1. HSAE cells were more susceptible than A549 cells to damage caused by direct contact with killed A. fumigatus germlings and by secreted fungal products. In response to A. fumigatus infection, A549 cells secreted a broader profile of cytokines and chemokines than HSAE cells. Taken together, these results demonstrate that studies of HSAE cells provide complementary data to A549 cells and thus represent a useful model for probing the interactions of A. fumigatus with bronchiolar epithelial cells in vitro. Importance During the initiation of invasive aspergillosis, Aspergillus fumigatus interacts with the epithelial cells that line the airways and alveoli. Previous studies of A. fumigatus-epithelial cell interactions in vitro used either large airway epithelial cell lines or the A549 type II alveolar epithelial cell line; the interactions of fungi with terminal bronchiolar epithelial cells were not investigated. Using the TERT-immortalized human small airway epithelial HSAEC1-KT (HSAE) cell line, we developed an in vitro model of the interactions of A. fumigatus with bronchiolar epithelial cells. We discovered that A. fumigatus invades and damages A549 and HSAE cell lines by distinct mechanisms. Also, the proinflammatory responses of the cell lines to A. fumigatus are different. These results provide insight into how A. fumigatus interacts with different types of epithelial cells during invasive aspergillosis and demonstrate that HSAE cells are useful in vitro model for investigating the interactions of this fungus with bronchiolar epithelial cells.

Multilayer Coating with Red Ginseng Dietary Fiber Improves Intestinal Adhesion and Proliferation of Probiotics in Human Intestinal Epithelial Models.

To exert their beneficial effects, it is essential for the commensal bacteria of probiotic supplements to be sufficiently protected as they pass through the low pH environment of the stomach, and effectively colonize the intestinal epithelium downstream. Here, we investigated the effect of a multilayer coating containing red ginseng dietary fiber, on the acid tolerance, and the adhesion and proliferation capacities of three Lactobacillus strains (Limosilactobacillus reuteri KGC1901, Lacticaseibacillus casei KGC1201, Limosilactobacillus fermentum KGC1601) isolated from Panax ginseng, using HT-29 cells, mucin-coated plates, and human pluripotent stem cell-derived intestinal epithelial cells as in vitro models of human gut physiology. We observed that the multilayer-coated strains displayed improved survival rates after passage through gastric juice, as well as high adhesion and proliferation capacities within the various gut epithelial systems tested, compared to their uncoated counterparts. Our findings demonstrated that the multilayer coat effectively protected commensal microbiota and led to improved adhesion and colonization of intestinal epithelial cells, and consequently to higher probiotic efficacy.