Infection and Immunity

Mycoplasma pneumoniae is a leading cause of community acquired pneumonia and bronchitis in people, particularly children and young adults. Like many bacterial pathogens, M. pneumoniae can grow in biofilm towers on both biotic and abiotic surfaces. Current models indicate that planktonic M. pneumoniae are simply individual cells that are dispersed from biofilm towers due to physical shear forces, and do not represent a bona fide planktonic growth state. However recent data indicate that these fractions may be physiologically distinct and may differentially influence disease outcome. We observed that experimental infection of mice with inoculum containing both adherent and non-adherent fractions of M. pneumoniae produced more severe lung pathology than inoculum prepared from M. pneumoniae adherent biofilms alone. Direct comparison of experimental infection with titer matched non-adherent (P-Mp) and adherent (B-Mp) fractions revealed that P-Mp produces more severe lung pathology compared to infection with B-Mp. Furthermore, experimental infection with a lower dose of P-Mp resulted in similar lung pathology relative to B-Mp infected mice. This increase in pathology was not associated with differences in bacterial burden, bacterial dispersal during inoculum preparation, nor due to differences in the phenotype of host immune responses. Comparative proteomics revealed that certain known and putative virulence factors associated with immune evasion are upregulated in P-Mp, potentially explaining the difference in virulence between the two fractions. Furthermore, comparative proteomics revealed distinct metabolic phenotypes between the two fractions, consistent with distinct planktonic and biofilm growth states, indicating that P-Mp likely represents a bona fide planktonic growth state. Collectively, these data implicate the growth state of M. pneumoniae as a determinant of in vivo virulence for this atypical human pathogen.

Background Group B Streptococcus (GBS) is a leading cause of bacterial sepsis globally. Late-onset (LO) GBS disease is preceded by colonization within the gastrointestinal tract in vulnerable neonates. Previous studies demonstrate that Candida albicans (C. albicans) promotes GBS colonization of host tissues including the bladder and vagina. However, its impact on GBS colonization of the intestinal tract is unknown. Methods We established a new animal model to study GBS and C. albicans co-colonization model in the context of the newborn host to examine C. albicans impact on GBS and further evaluated fluconazole prophylactic treatment to these same outcomes. Physical interactions between organisms in vivo were imaged and GBS gene expression was evaluated. Results C. albicans increased GBS burden in the neonatal gut and GBS was physically colocalized with C. albicans hyphae as evidenced by the colocalization analysis. Fluconazole treatment of co-colonized mice decreased extraintestinal GBS dissemination. C. albicans presence upregulated genes encoding proteins responsible for GBS adhesion and invasion of intestinal epithelial cells. Conclusion C. albicans enhances GBS burden and pathogenicity in the neonatal gut. These findings highlight the need for further investigation into C. albicans as a potential risk factor for GBS colonization and LO disease in human infants.

Innate immune cells, including macrophages and DCs, are critical for controlling infection by Cryptococcus neoformans. Although C. neoformans is phagocytosed equally by each pulmonary phagocyte subset, CD11b+ DCs support fungal growth, whereas Ly6c- monocyte-like macrophages are antifungal. The metabolic programs underlying these contrasting responses remain incompletely understood, but our previous work points to increased glycolysis in antifungal cells. Therefore, we hypothesized that metabolic reprogramming in phagocytes following cryptococcal exposure leads to different outcomes. We examined ten metabolic pathways using flow cytometry, metabolic inhibition, antifungal assays, and pulmonary infection. Cryptococcal exposure increased GLUT-1, HK, HIF-1, and lactate production in J774A.1 macrophages, consistent with enhanced glycolytic metabolism in antifungal cells. In contrast, ATP5a and IDH2 were reduced, suggesting diminished mitochondrial metabolism. Cryptococcus also decreased G6PD, ACAC, ASS1, and PRDX2, indicating alterations in PPP activity, fatty acid synthesis, arginine metabolism, and antioxidant defense. Pharmacologic inhibition of PPP further reduced G6PD expression and significantly decreased intracellular cryptococcal burden, supporting a role for glucose-derived metabolic pathways in regulating fungal survival. In vivo, infection increased GLUT-1 and HK expression in antifungal female Ly6c- monocyte-like macrophages but not in permissive CD11b+ DCs, whereas other metabolic markers showed limited or subset-specific changes. Collectively, these findings demonstrate that antifungal cells use a predominantly glycolytic program accompanied by reduced mitochondrial, anabolic, and antioxidant-associated protein expression following interaction with C. neoformans. This phagocyte subset-dependent metabolic response during pulmonary infection may influence antifungal activity and identify metabolic pathways that could be targeted to strengthen host defense.

Clostridioides difficile infection (CDI) is characterized by toxin-mediated epithelial injury, intestinal inflammation, and a high frequency of disease recurrence. Although complement components are produced locally within the gastrointestinal tract, their contribution to C. difficile pathogenesis remains poorly understood. Here, we investigated the interaction of the complement proteins C1q and C3 with C. difficile spores and their contribution to spore-host interactions and disease outcome. We found that C1q and C3 are accessible within the healthy ileal mucosa and that TcdB intoxication differentially remodels their spatial availability, decreasing accessible C1q while increasing accessible C3 at higher toxin concentrations. C. difficile spores associated with both complement components in vivo and directly interacted with purified human C1q and C3 in vitro, with immunogold electron microscopy localizing these interactions to the outer exosporium. Although the collagen-like BclA proteins modulated complement binding under some conditions, they were not required for C1q or C3 association. Far-Western analysis coupled to mass spectrometry identified the exosporium proteins CotE and CdeM as candidate complement-interacting proteins, and purified CdeM inclusion bodies directly associated with both C1q and C3. Exposure of spores to human serum promoted their interaction with intestinal epithelial cells, whereas depletion of either C1q or C3 markedly reduced spore internalization and reconstitution with the corresponding purified protein partially restored entry. C3-derived species, including C3dg, were selectively associated with spores but not vegetative cells, without reducing spore viability. Finally, C1q deficiency did not alter the initial onset of CDI but accelerated recovery and markedly attenuated recurrent disease following vancomycin treatment. Together, these findings identify intestinal complement as a previously unrecognized component of the C. difficile spore-host interface and reveal C1q and C3 as modulators of spore epithelial entry, persistence, and recurrent disease.

Metals are essential for all forms of life and are therefore the subject of tight competition at the host-pathogen interface. Manganese (Mn) is an important cofactor for enzymes involved in various processes in bacteria, including the response to oxidative stress, and the ability to scavenge Mn through the expression of high-affinity Mn transporters has been associated with virulence of many extracellular and vacuolar pathogens. While the cytosol has been established to be restrictive for growth of non-adapted pathogens, the extent to which metal limitation contributes to this is not well understood. Here, we investigate whether high-affinity Mn acquisition is important for intracellular adaptation and virulence of the cytosolic pathogen Listeria monocytogenes (Lmo). To do so, we generated a strain of Lmo in which both of its predicted high-affinity Mn transporters, MntH and MntABC, are disrupted. We found that this strain was severely defective in growth in vitro under Mn-limited conditions and had lower intracellular Mn content. Additionally, we observed Mn-dependent transcriptional regulation of the mntH and mntA genes. Together, these data strongly support that MntH and MntABC are high-affinity Mn transporters and are likely to be the only such dedicated Mn transporters expressed in Lmo. Using the {Delta}mntH {Delta}mntA strain, we provide evidence that Mn limitation sensitizes Lmo to oxidative stress in vitro, consistent with the known role of Mn in mitigating reactive oxygen species. Ultimately, we demonstrate that expression of high-affinity Mn transporters is essential for intracellular replication of Lmo in macrophages, plaquing on fibroblast monolayers, and for virulence in an animal model of systemic infection. Our work, therefore, suggests that Lmo is challenged by Mn limitation in the cytosol and requires Mn scavenging to adapt to its intracellular niche.

Chlamydia trachomatis is an obligate intracellular, developmentally regulated pathogen with a reduced genome as a consequence of adapting to the host cell environment. Despite possessing only eight transcription factors, its developmental cycle is tightly regulated. Hence, we hypothesize that factors like post-transcriptional and post-translational modifications play significant roles in controlling developmental progression. Chlamydia encodes serine/threonine kinases, including an ortholog of Pkn1. In this study, we characterized the function of Pkn1 in chlamydial biology and revealed its role as a cell division regulator. We designed an anhydrotetracycline (aTc)-inducible construct encoding Pkn1 tagged with six histidines at the C-terminus (Pkn1_6xH). Wild-type Pkn1 localized primarily at the division septum whereas a kinase-null Pkn1 isoform localized in the cytosol. To further explore the association of Pkn1 with the divisome, we designed a plasmid encoding an aTc-inducible CRISPRi system targeting pkn1(pkn1KD). Intriguingly, decreased pkn1 transcript levels resulted in enlarged chlamydial cells with irregular coccoid shapes akin to penicillin-treated cells, indicating a block in cell division when pkn1 transcripts are reduced. These effects were complemented by expressing an additional wild-type copy, but not a kinase-null copy, of pkn1 during knockdown. Using a bacterial two hybrid system, we identified an interaction between Pkn1 and MreB, a cytoskeletal protein forming a ring at the septum in Chlamydia. We showed the co-localization of these proteins in Chlamydia and that inhibition of MreB function disrupts Pkn1 localization. Overall, our data indicate an essential function for Pkn1 activity in regulating cell division in Chlamydia.

Bacillus cereus biovar anthracis (Bcbva) strains, which cause fatal anthrax-like disease, have been isolated from animals in Central and West Africa. Like B. anthracis, Bcbva produces a poly-{gamma}-D-glutamic acid (PDGA) capsule, as well as edema factor (EF), lethal factor (LF) and protective antigen (PA). Bcbva also possesses a hyaluronic acid capsule that is not expressed by B. anthracis. The pathogenesis of Bcbva has recently been characterized in inhalational infection models, in rabbits and guinea pigs. Here, we characterized the ki-netics of Bcbva infection and toxin production in a murine model of cutaneous infection using the Bcbva CA strain isolated in Cameroon. We found that spore germination oc-curred rapidly at the inoculation site, followed by bacterial dissemination to the draining cervical lymph nodes. Germination was associated with rapid production and diffusion of LF and EF. Bacteria rapidly disseminated to lymphoid organs and other compartments, with distal germination observed 18 hours post-infection. Infected mice developed septi-cemia within 24 hours, accompanied by high circulating LF levels and signs of cardiac and renal injury. These findings may contribute to the fulminant course of Bcbva infection observed in animals and highlight its potential risk to humans.

Vibrio cholerae, the facultative pathogen underlying cholera, naturally inhabits warm aquatic estuaries. Environmental persistence is enhanced by the ability of V. cholerae to colonize host reservoirs and form multicellular biofilms, causing seasonally endemic outbreaks in many tropical regions. Most toxigenic strains utilize the type IVa mannose-sensitive hemagglutinin (MSHA) pilus for host reservoir colonization and biofilm formation. Temperature and salinity can alter V. cholerae biofilm formation, yet their impact on MSHA production specifically remains largely unknown. Here, we utilized transcriptional reporters of predicted msh promoters (msh-P1/msh-P2/msh-P3) and functional assays, to determine temperature and salinity impacts on msh expression and pilus biogenesis. Under standard laboratory conditions (30{degrees}C, 1% NaCl) only msh-P1/P2 are active and inversely-coordinated with one another. Both msh-P1/P2 activity were elevated by high temperature (37{degrees}C) and low salinity (0.25%/0.5% NaCl), and reduced by low temperature (20{degrees}C/25{degrees}C) and high salinity (2%/3% NaCl). Temperature-mediated alterations in promoter activity were not immediately reflected in changes to cell-surface MSHA levels, whereas high salinity led to decreased MSHA production. Combining high temperature (37{degrees}C) and high salinity (2%/3% NaCl), attenuated the salinity-mediated reduction of msh-P1/P2 activity. Biofilm biomass levels were only substantially heightened at 25{degrees}C and 20{degrees}C, likely a result of no temperature-dependent changes in cell-surface MSHA, and additional temperature-controlled biofilm regulation previously described. We also found msh-P1/P2 promoter activity and MSHA production varies widely across toxigenic O1 and O139 serogroups despite complete sequence homology. These results shed new light on how key signals regulate MSHA pilus production to support V. cholerae persistence in aquatic environments.

Coxiella burnetii ( Cb ), the causative agent of Q fever, replicates within host macrophages by modulating innate immune responses through its type IVB secretion system (T4SS). Host cells sense pathogens via Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), to recognize pathogen-associated molecular patterns (PAMPs) and initiate signaling pathways that drive pro-inflammatory cytokine and interferon responses. Toll-like receptor 3 (TLR3) triggers the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and Interferon regulatory factors (IRFs) resulting in pro-inflammatory cytokine production and type I interferon (IFN-I) induction. Here we demonstrate that Cb requires T4SS to suppress TLR3-induced NF-{kappa}B and IRF transcriptional responses. Furthermore, RNA purified from both virulent and avirulent Cb is sufficient to activate TLR3, identifying pathogen RNA as a relevant PAMP in this context. Using a pulmonary infection model with virulent Cb, we found that TLR3/ Toll-Interleukin-1 Receptor Domain-Containing Adapter Protein Inducing Interferon Beta (TRIF)-dependent innate immune pathway inhibits cachexia. In contrast, signaling through the interferon-/{beta} receptor (IFNAR) restricts bacterial dissemination, indicating that these pathways play distinct but complementary roles in host defense. Mechanistically, Cb suppresses TLR3/TRIF signaling in a T4SS-dependent manner by preventing host TLR3 recruitment to the Coxiella-containing vacuole (CCV) and disrupting TRIF-TNF Receptor-Associated Factor 6 (TRAF6) interactions, thereby selectively inhibiting impairing NF-{kappa}B activation. Consistent with this, we identify five T4SS effector proteins that attenuate TLR3-induced NF-{kappa}B signaling, including CBU1292, which suppresses both NF-{kappa}B and IRF-dependent responses downstream of TLR3.

Disruption of the intestinal microbiota increases susceptibility to Clostridioides difficile infection (CDI), creating an opportunity for prophylactic interventions before symptomatic disease develops. We investigated whether Escherichia coli Nissle 1917 (EcN) engineered to express the bacteriophage-derived endolysin CD27L could enhance pathogen inhibition and attenuate CDI. Using a plasmid-cured EcN chassis and pMUT2-derived vectors, we generated strains expressing CD27L under constitutive or anaerobically regulated promoter control. The constitutive strain, BioEcN1, reduced viable C. difficile recovery relative to parental EcN and was selected for animal studies. BioEcN1 remained recoverable from monoassociated mice without in vivo antibiotic selection, with similar recovery on selective and nonselective media supporting construct retention. Prophylactic administration reduced clinical disease, body-weight loss, and intestinal histopathological injury in germ-free mice. Observed survival was 100% in BioEcN1-treated challenged mice versus 50% in untreated challenged mice during the monitored period. Protection was accompanied by lower fecal and cecal pathogen burdens. In mice harboring a human-derived dysbiotic microbiota, prophylactic BioEcN1 also reduced clinical and stool-consistency scores, with lower early fecal pathogen recovery and less colon shortening. These findings establish preclinical proof of concept for prophylactic administration of an endolysin-expressing probiotic to attenuate CDI.

ABSTRACT Background: Trachoma remains the leading infectious cause of blindness. Over 100M people are at risk, and 1.9M already live with visual impairment or blindness. Ethiopia bears the greatest burden of disease with ~61M or 59% of the known affected global population. Although Chlamydia trachomatis (Ct) infection initiates disease, infection alone fails to explain why inflammation resolves in some but progresses to irreversible scarring and vision loss in others. Previous studies have largely examined gene expression or limited protein panels, leaving the conjunctival immune protein environment poorly characterized. We hypothesized that distinct conjunctival immune states capture these pathogenic processes and could reveal biomarkers for selecting treatment and surveillance strategies. Methods and Findings: We conducted a cross-sectional study of 667 participants residing in the hyperendemic Amhara region of Ethiopia at least one year after the most recent azithromycin mass drug administration to treat Ct. The upper tarsal conjunctivae was graded for TF and TI, trachomatous inflammation-follicular or intense, respectively; I-TS/TT, inflammation with scarring with or without trichiasis, defined as one or more eyelashes touching the globe of the eye; and TS and TT, scarring with or without trichiasis but no inflammation. T0 represented no trachoma. The protein concentrations of 41 immune mediators were quantified using Meso Scale Discovery electrochemiluminescence immunoassays. Associations with infection and grade were evaluated using covariate-adjusted models with false discovery rate correction. Bernoulli mixture modeling (BMM) revealed four distinct Immune State Types (ISTs). Both approaches supported a shared biological framework characterized by selective immune activation in Ct infection, broad inflammation in TF/TI, and chemokine remodeling versus immune quiescence in the scarred conjunctiva. Ct infection was associated with a focused IFN-{gamma}-centered response. IST1 captured this immune response where 51.9% of the participants had Ct; we therefore termed IST1 Quiescent Infection. In contrast, TF/TI was associated with higher concentrations of proinflammatory, Th1/Th17, chemokine, regulatory, and remodeling pathways mediators. This profile fit well within IST2, termed Broad Inflammation, where 72.4% of the participants had TF/TI. TS/TT retained considerable chemokine activity, whereas I-TS/TT showed a narrower, IL-17A-dominant inflammatory profile. BMM separated scarring into two states characterized by distinct profiles: IST3, Conjunctival Remodeling, with significantly stronger chemokine and T-cell maintenance signals; and IST4, Quiescent Scarring, with the majority of chemokines being significantly downregulated. This suggests that scarred conjunctiva can retain substantial immune activity or shift toward a more attenuated state. Conclusions: This study establishes the first IST framework for trachoma, revealing biologically distinct immune states with different potential clinical implications. IST1 indicates the need for antibiotic treatment, whereas IST2 suggests that novel immunotherapeutics might be effective in thwarting disease progression. The upregulated chemokines in IST3 may indicate risk of progression to TT necessitating prospective studies and potential novel antifibrotic interventions. Together, our findings provide a strong biological foundation for stratifying patients by immune state and matching them to preventive and therapeutic care. Prospective validation and streamlined biomarker panels will support the translation of this framework into field settings to help avert disease progression and blindness.

Cytolysin is a toxin-bacteriocin commonly encoded by Enterococcus faecalis that can target erythrocytes, neutrophils, and macrophages as well as other Gram-positive bacteria. Cytolysin can lyse human, horse, rabbit, and mouse erythrocytes, but not sheep erythrocytes (SEs). It is hypothesized that SEs are resistant to cytolysin because they lack phosphatidylcholine (PC) in their membranes, while susceptible erythrocytes have PC-rich membranes, suggesting PC as a cytolysin binding target. Here, we isolated a urinary E. faecalis strain C33 that can efficiently lyse SEs and found that this activity was conserved among several E. faecalis clinical isolates. We demonstrated that although C33 cytolysin was necessary for SE lysis, it was not sufficient, suggesting a secondary factor enhances cytolysin activity against SEs. Through liposome assays we found that while PC was required for the activity of DS16 cytolysin, PC was dispensable for C33 cytolysin activity. To assess the biological significance of C33 cytolysin in the bladder microenvironment, we assessed C33 cytolysin activity against bladder epithelial cells and the urinary microbiota. We found that C33 cytolysin was more cytotoxic to human bladder epithelial cells than the canonical DS16 cytolysin. We also found greater inhibition of co-isolated urinary microbiota species Streptococcus parasanguinis, Enterococcus raffinosus, Staphylococcus capitis, and Corynebacterium coyleae by C33 cytolysin. Interestingly, E. faecalis C33 also inhibited the protective urogenital taxa Lactobacillus crispatus and Bifidobacterium longum. Together, these results reveal a new PC-independent cytolysin activity among clinical E. faecalis isolates that may support survival and competition in the urinary microenvironment.

Serious pertussis infections disproportionately affect infants but the biological basis for this age-dependent susceptibility remains unclear. Infant mouse models recapitulate features of severe human infant pertussis. We investigated the role of interferon lambda (IFN-{lambda}), a key regulator of mucosal immunity, in Bordetella pertussis infection of infant mice. While infected adult mice upregulate lung IFN-{lambda}, infant mice inoculated at P7 fail to upregulate IFN-{lambda} and succumb to infection. We hypothesized that failure to produce IFN-{lambda} during infection represents a critical immunological deficit in infant mice, and that restoring IFN-{lambda} signaling would improve survival outcomes. Whereas wild-type mice gained complete protection from lethal B. pertussis infection by P10, mice lacking the IFN-{lambda} receptor component IFNLR1 did not achieve full protection until P21. Loss of IFNLR1 was associated with enhanced bacterial dissemination to systemic organs. Infant mice possessed a functional IFN-{lambda} receptor in the lungs but failed to upregulate IFN-{lambda} during infection, and supplementing IFN-{lambda} exogenously extended survival. RNA sequencing of lung tissue from infected and uninfected wild-type and IFNLR1-deficient mice inoculated at different ages identified an immune transcriptional framework distinguishing susceptible from resistant animals at a systems level and revealed IFN-{lambda} signaling as a critical driver of immunological maturation in the infant lung. Infected infant IFNLR1-deficient mice had dysregulated immune cell recruitment to the lungs, indicating a quantitatively expanded but qualitatively impaired response. These findings demonstrate that IFN-{lambda} affects immune maturation accounting for a critical window of age-dependent resistance to lethal pertussis with novel therapeutic possibilities for human infants with this disease.

Cholera toxin (CTx) is a potent enterotoxin and widely used mucosal adjuvant, yet the acute host response to CTx itself, in the absence of a co-administered antigen, remains vague. In this study, we integrated physiological and metabolic phenotyping with intestinal transcriptomics, epithelial imaging, immune profiling, hepatic gene-expression analysis, and paired microbiome profiling to characterize the response to a single oral CTx exposure in mice. Within 24 h, CTx remodeled immune-cell composition in both epithelial and lamina propria compartments, with responses shaped by sex and genetic background. Recurrent changes included increased epithelial group 3 innate lymphoid cells, reduced lamina propria ROR{gamma}t+ regulatory T cells, and decreased epithelial ILC1/NK cells in males of both strains. CTx also increased villus DCLK1+ tuft cells and reduced MUC2 immunoreactivity. Jejunal transcriptomics revealed induction of inflammatory defense, glycolytic, autophagy, and lipid-utilization programs together with suppression of proliferative and mitochondrial programs, consistent with coordinated stress adaptation. These local changes were accompanied by rapid restructuring of the fecal microbial community and by systemic hypometabolism, despite markedly different hypothermic responses across strains. CTx also elicited a selective hepatic stress and metabolic program, including conserved suppression of Cyp7a1 across sexes and strains. Together, these findings show that CTx alone rapidly reorganizes the intestinal immune, epithelial, metabolic, and microbial environment while suppressing whole-body metabolism. This work provides an integrated framework for understanding the acute host response to CTx and for interpreting its widespread use as a mucosal adjuvant.

Pre-prolipoprotein phosphatidylglycerol diacylglyceryl transferase (Lgt) catalyzes the first step in the lipoprotein modification pathway and is essential for growth and viability of Helicobacter pylori. We explored the fate of H. pylori cells under Lgt depletion conditions to obtain insight into Lgt function on morphology and physiology. Downregulation of lgt expression led to growth arrest and a heterogenous population of small rod-shaped cells and small round cells with spiky protrusions, a loose outer membrane and disintegrated flagella. H. pylori overcomes a defect in lipoprotein modification through alteration of its gene expression profile resulting in so-called revertants. Outer membrane protein synthesis, carbon metabolism and nutrient uptake was reduced, and protein translation and flagellar biogenesis increased. Lgt specific regulation was observed in phospholipid biosynthesis resulting in an initial increase in cardiolipin followed by an increase in lysophospholipids at the time of adaptation. Suppressors of {Delta}lgt that were selected on solid media under restrictive conditions either lacked chromosomal mutations or had mutations in the cagUT-tetR regulon controlling the expression of lgt. A suppressor without mutations had an altered expression profile that led to downregulation of a DNA methyltransferase and upregulation of HP_0014, a motility associated glycosyltransferase family protein. Transmission electron microscopy and single cell video microscopy showed that a subpopulation of cells is motile in the revertant while the suppressor regained full motility with higher average speed compared to the wild type strain. Overall, our data show that lipoprotein modification by Lgt is important for membrane and flagellar integrity and that the heterogenous character of H. pylori cultures allows individual cells to overcome this cell envelope stress and survive. Our findings are important for antibacterial development strategies targeting essential integral membrane enzymes involved in lipoprotein modification and stimulate exploration of synergetic activity between known antibiotics and novel antibacterial agents targeting novel cellular pathways.

Aspergillus fumigatus is the most common cause of invasive aspergillosis (IA), an opportunistic pulmonary infection of immunocompromised patients that can be fatal, despite treatment with modern antifungal drugs. During infection, myeloid cells, such as neutrophils, monocytes, and monocyte-derived dendritic cells (Mo-DCs) are recruited to the lung and are critical for phagocytosing and killing A. fumigatus spores. Recent evidence has shown that intracellular crosstalk between these professional immune cells and other resident pulmonary cell types is required for an effective immune response to A. fumigatus, and that signaling via the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is part of this intracellular communication. In this study, we sought to determine a role for GM-CSF signaling on monocytes and their progeny. We harnessed a mouse model of deletion of GM-CSF receptor {beta} chain (encoded by Csf2rb) in CCR2-expressing cells. Using this model, we found that GM-CSF signaling is required on CCR2+ cells for killing of A. fumigatus and for host survival. This observation expands the importance of GM-CSF in host defense during A. fumigatus pulmonary infection and extends its role in pathways of intracellular crosstalk that mediate effective pulmonary antifungal immunity.

Little is currently known about the regulation of PL fimbriae produced by some pathogenic E. coli strains. We identified PlfB, a transcriptional regulator of the PapB family, as a negative regulator of PL fimbriae (Plf). Deletion of plfB significantly increased plfA transcription and Plf production. PlfB bound specifically to the plfA promoter region. Using a PplfA-lux reporter system, the deletion of plfB caused sustained and increased expression across serial passages. Global regulatory genes such as lrp, argR, and hns were also implicated in regulation of plf expression. Loss of lrp or argR decreased plf expression, suggesting these regulators positively regulate PL fimbriae. By contrast, loss of hns increased levels of plf expression, supporting its role as a negative regulator. Interestingly, loss of plfB significantly increased bacterial colonization in the lung of 6-day-old turkey poults. Further, complementation of plfB restored plf fimbrial expression and colonization of turkey lungs to wild-type levels. This further supports an important role for PL fimbriae for colonization of turkey lungs, since loss of plfB which resulted in increased plf gene expression in turkey lungs also correlated with higher bacterial numbers in the lungs. Taken together, these results indicate that PlfB acts as a negative regulator to reduce levels of PL fimbrial expression, under some conditions, but that it is not required for plf expression. This contrasts with the role the PapB regulator, which has been shown to play an essential role in expression of P fimbriae in uropathogenic E. coli strains.

Adherent Invasive Escherichia coli (AIEC) colonize the ileal mucosa of Crohn's disease patients. Among bacterial virulence factors potentially implicated, type 6 secretion systems (T6SS) are molecular machineries sending effectors in prokaryotic or eukaryotic target cells. Two T6SS have been reported in AIEC LF82 reference strain. Here, we show that T6SS genes are more prevalent in Crohn's disease patients' microbiota compared to healthy controls, and that T6SS are more frequent in AIEC pathovar compared to E. coli in general and associated with FimH pathoadaptive polymorphism. The use of an in vitro gastrointestinal system coupled with T6SS promoter reporter assay highlighted expression of LF82 T6SS under gastrointestinal conditions, with a major role for bile salts. In mice infected with AIEC LF82 versus T6SS-deleted mutants, T6SS showed a functional impact in modulating the composition of mucosa-associated gut microbiota. Thus, AIEC may use their T6SS to compete with endogenous microbiota to colonize host mucosa.

Shigella flexneri is an enteric pathogen responsible for causing shigellosis, a type of bacillary dysentery of significant global health concern. The increasing prevalence of antibiotic resistance has driven the search for alternative treatments, including bacteriophage (phage) therapy. However, the effectiveness of orally administered phages for enteric pathogens depends on their ability to survive passage through the gastrointestinal tract. In this study, the ability of bacteriophage Sf14 to maintain infectivity was evaluated under simulated gastrointestinal conditions using a modified INFOGEST 2.0 in vitro digestion model. This model includes salivary, gastric, and intestinal fluids with physiologically relevant compositions of electrolytes, bile salts, and enzymes. Phages were incubated in each simulated phase at standard physiological temperature, then tested for infectivity using quantitative plaque assays. Results show that Sf14 remained stable during the short duration of the oral phase and for the duration of the intestinal phase. By contrast, survival in simulated gastric fluid required a pH of 2.75 or higher, with rapid and substantial loss of infectivity at pH 2.5 and below. These findings demonstrate that while Sf14 is stable in oral and intestinal environments, the gastric phase poses a critical barrier to phage survival. With in vivo fasting gastric pH ranging from 1.5 to 3, encapsulation would enable the most flexible oral phage therapy against enteric pathogens, but ingestion after eating or alongside antacids may be sufficient.

Mycobacterium tuberculosis has a flavin-intensive metabolism, with more than 180 proteins that require flavin or deazaflavin cofactors. M. tuberculosis requires riboflavin biosynthesis for growth in standard culture conditions in vitro, which can be overcome by providing exogenous riboflavin. It is unknown whether M. tuberculosis also requires riboflavin synthesis to grow or persist in infected host cells or tissues. Here we used a M. tuberculosis strain that conditionally expresses ribA2, which encodes a bifunctional enzyme that catalyzes the first steps in both branches of riboflavin biosynthesis, to determine whether M. tuberculosis requires riboflavin biosynthesis during infection. Transcriptional repression of ribA2 caused death of M. tuberculosis in vitro without riboflavin supplementation. RNAseq revealed that riboflavin starved M. tuberculosis induce a transcriptional program indicating decreased electron transport chain function and broad changes to central metabolism. Transcriptional repression of ribA2 prevented M. tuberculosis growth in cultured macrophages and caused clearance of M. tuberculosis from infected tissues at all stages of infection in both C57BL/6 mice that form cellular lung lesions and C3HeB/FeJ mice that develop necrotic lung granulomas. Our results demonstrate that M. tuberculosis requires RibA2 for viability at all stages of infection and support further characterization of the riboflavin biosynthesis pathway as a potential target for development of new anti-tubercular drugs.