Infection and Immunity

Following an intravaginal inoculation with Chlamydia trachomatis, mice deficient in type I interferon receptor IFNR1 (IFNR1-/-) significantly increased the yield of live chlamydiae on days 3 & 5 but reduced it to the level of wild-type mice by day 7, while mice deficient in type II interferon receptor IFN{gamma}R1 (IFN{gamma}R1-/-) significantly increased the chlamydial yield by day 5 and the increase persisted throughout the remainder of the infection course. These observations reveal a temporal division of labor between type I & II interferons in regulating C. trachomatis infection in the female genital tract. Interestingly, mice deficient in both IFNR1 & IFN{gamma}R1 exhibited higher mortality and shed more chlamydial organisms than IFN{gamma}R1-/- mice by week 6, suggesting that IFNR1 remains critical for inhibiting C. trachomatis at late stages. An anti-IFNR1 antibody blockade significantly increased chlamydial yields in IFN{gamma}R1-/- mice, suggesting that the anti-chlamydial activities of type I & II interferon systems are both distinct and overlapping throughout the infection course. Furthermore, the anti-chlamydial activity of type I interferon signaling is localized to the lower vagina, while that of type II interferon signaling is localized to the upper vagina. Thus, we have demonstrated that type I & II interferons function complementarily and synergistically in time and space to control C. trachomatis infection, laying the foundation for further elucidating the mechanisms of IFN regulation of chlamydial infection and for developing interventional and preventive strategies against C. trachomatis in the female genital tract. ImportanceLack of information on the precise roles of type I and type II interferons during chlamydial infection has hindered the development of interferon-based strategies to prevent chlamydial infection and pathogenicity. The current study has revealed a temporal division of labor between type I & type II interferons in regulating chlamydial infection in the female genital tract, with type I acting earlier than type II during the innate phase. Nevertheless, type I remains critical for cooperating with type II to suppress chlamydia 6 weeks after infection. Finally, type I interferons seem to mainly target chlamydial infection in the lower vagina, while type II interferons target the infection in the upper vagina. These new findings on the distinct and overlapping roles of type I and type II interferons in regulating chlamydial infection may guide the development of interferon-based precision strategies to reduce chlamydial infection and pathogenicity in the female genital tract.

Previous research demonstrated that Campylobacter jejuni encodes a heme utilization system that facilitates heme-dependent growth under iron-limiting conditions and that transcription of this system is induced during human infection. Despite these observations, it remained unknown whether the heme transport system is required for colonization and disease in a susceptible host. To address this, we created individual non-polar deletion mutants of each component of the heme transport system and examined their ability to promote heme-dependent growth and iron uptake. From this work, we found that only the heme receptor, ChuA, was required for heme-dependent growth and iron acquisition, which supports the earlier work of another group. Further, we examined whether intestinal colonization, immune activation, and pathology were altered during infection with these mutants. After establishing that elevated heme and chuABCD expression occur during C. jejuni infection of IL-10-/- mice, we found that a mutant of the heme receptor, ChuA, exhibited significantly reduced colonization of the colon. In addition, we found that neutrophil and circulating monocyte recruitment were significantly reduced in the colon during infection with the ChuA mutant, but that populations of self-maintaining tissue macrophages remained high. Loss of ChuA reduced colonic colonization and was accompanied by diminished innate immune cell recruitment and intestinal pathology. Together, these findings identify ChuA-dependent heme acquisition as a key determinant of efficient colonic colonization and the associated inflammatory disease.

The phylum Chlamydiota comprises obligate intracellular bacteria characterized by a highly conserved, biphasic developmental cycle. This cycle involves the transition between the infectious, metabolically quiescent elementary body (EB) and the non-infectious, replicative reticulate body (RB). While the morphological transitions of the developmental cycle are well-documented, the regulatory mechanisms governing these phenotypic shifts remain poorly understood. A primary candidate for this regulation is Euo, a conserved, phylum-specific helix-loop-helix transcription factor hypothesized to repress late-cycle genes and prevent premature differentiation. In this study, we employed CRISPR interference (CRISPRi) to knockdown euo expression in Chlamydia trachomatis to further elucidate its role in developmental regulation. Unexpectedly, euo knockdown did not significantly disrupt the primary developmental cycle; progression through RB replication, the formation of intermediate bodies (IBs), and the kinetics of late-gene expression remained largely comparable to wild-type. However, we observed a significant reduction in the production of infectious progeny. Detailed analysis revealed that while EBs were still produced and capable of entering host cells after knock down of euo, these EBs exhibited dysregulated gene expression during the germination phase of a new infection cycle. Consequently, these bacteria failed to establish a productive secondary infection. These results suggest that rather than acting as a developmental switch for differentiation during the initial infection, Euo is essential for the proper programming of EBs, ensuring transcriptional competence upon re-infection of a host cell.

Coxiella burnetii alternates morphologies to survive in two niches: the external environment and a degradative intracellular compartment. The small cell variant (SCV) is adapted for environmental persistence and transmission of Q fever to ruminants and humans. The large cell variant (LCV) is intracellular, and despite not being a major source of transmission, is infectious in vitro. When modeling infection, researchers typically apply a mixed population of these cell types as inocula. As this practice does not mimic natural infection, it may confound our understanding of early Q fever infection events. We separated SCV and LCV by density gradient centrifugation and compared their replication in primary murine macrophages and a fibroblast cell line. SCV inocula replicated more efficiently than LCVs in both host cell types. LCV replication was delayed for four days in macrophages compared with SCV inocula, which had completed logarithmic growth by that time point. We found no difference in pathogenic vacuole size, but there was a modest difference in their respective bacterial burdens. Interestingly, IL-6 and CXCL2 secretion was significantly elevated in LCV-infected macrophages as compared to SCV at 24 hours, suggesting a difference in the host response to each. This is the first study to demonstrate that C. burnetii developmental status influences the progression of infection.

Platelet Factor 4 (PF4), a cationic antimicrobial peptide, serves as a ligand for the myeloid-specific phagocytic receptor CR3 (Mac-1, CD11b/CD18). We previously demonstrated that recombinant dimeric PF4 (rdPF4) functions as a bacterial opsonin, enhancing phagocytosis of Gram-positive Staphylococcus aureus and facilitating clearance of both antibiotic-susceptible and methicillin-resistant S. aureus in a mouse model of infectious peritonitis. In this study, we examined whether rdPF4 is pathogen-agnostic by assessing its effect on phagocytosis of Gram-negative encapsulated Klebsiella pneumoniae, a WHO Bacterial Priority Pathogen. We demonstrate that rdPF4 enhances CR3-mediated phagocytosis of both live and heat-inactivated high-virulence K2 and low-virulence K3 strains of K. pneumoniae by various mouse and human macrophage cell lines, as well as primary neutrophils and macrophages. It also increased phagocytosis of carbapenem-resistant K. pneumoniae. rdPF4 did not directly kill bacteria but acted as an opsonin binding to the negatively charged bacterial capsule and creating recognition sites for CR3 on leukocytes. In a mouse sepsis model, a single dose of rdPF4 significantly enhanced bacterial clearance from the lungs, liver, and peritoneum and reduced bacteremia. Histological analyses showed that rdPF4 provided substantial protection to lung and liver tissues against K. pneumoniae-induced damage. Consistent with these findings, rdPF4 treatment increased the survival rates of infected mice. These results show that rdPF4 effectively targets the capsule, a key virulence factor of K. pneumoniae, thereby reducing the bacteriums ability to evade the host immune response. Overall, the data suggest a common mechanism in which cationic rdPF4, by binding to the negatively charged surfaces of both Gram-negative and Gram-positive bacteria, diminishes their antiphagocytic properties.

Hemochromatosis and hemosiderosis are iron overload disorders that cause immune dysfunction and increase susceptibility to bacterial infections. There have been numerous case studies reporting septic-like outcomes for hemochromatosis patients infected with enteric Yersiniae; however, research regarding hemosiderosis and Yersinia infection is limited. Here, we have established a mouse model of hemosiderosis by feeding C57BL/6 mice a high-iron diet. These mice exhibit several indicators of iron overload that are seen clinically, including elevated serum iron levels and iron deposition in various tissues. Characterization of the iron overload mouse model shows that a high-iron diet induces local inflammation in the small intestine and systemic inflammation in a time-dependent manner. Oral infection with Yersinia enterocolitica causes complete mortality in the iron-overloaded mice, while wild-type mice all survive and effectively clear the infection. Lastly, we have observed that iron chelation therapies such as Deferoxamine and Deferisarox are detrimental to iron-overloaded mice during Yersinia infection. This work provides a model to further study iron overload disorders and Yersinia infection.

Periodontal disease is a chronic inflammatory condition that develops in response to oral microbiome dysbiosis and host-microbiome immune response dysregulation. The innate immune system plays a major role in the development and persistence of disease in part by producing inflammatory cytokines. One of the major cytokines implicated in disease is interleukin-1{beta} (IL-1{beta}), which requires inflammasome activation. Much of the oral microbiome, including Streptococci, which are otherwise considered commensal, is required for the full development of periodontal disease. We have previously reported that inflammatory-activated macrophages and neutrophils counterintuitively allow survival of internalized Streptococcus gordonii over non-activated phagocytes. This internal bacterial survival leads to inflammasome activation via the cytoplasmic activator NLRP6, but not NLRP3, and subsequent increases in IL-1{beta} release. Here, we test and find that the keystone pathogen Porphyromonas gingivalis can activate macrophages in a manner that allows for increased S. gordonii survival and IL-1{beta} production above levels when P. gingivalis interacts with macrophages alone. We also use the mouse ligature-induced periodontal disease model to test the importance of NLRP6 in disease development. We found mice lacking NLRP6 had significantly reduced bone loss, IL-1{beta}, and neutrophil infiltration following disease induced by P. gingivalis when S. gordonii or other mouse commensals were present, but had no effect when S. gordonii was inoculated alone. This work thus reveals an additional important inflammasome activation mechanism by which oral keystone pathogens may stimulate periodontal disease progression. Author SummaryChronic inflammation is a driver of many diseases, including periodontal disease. Periodontal disease is a long-lasting inflammatory disease caused by an unhealthy imbalance in the oral microbiome and an abnormal immune response toward those bacteria. A major inflammatory molecule involved in this inflammation is IL-1{beta}, which is produced after inflammasome activation. We previously found that immune cells such as macrophages and neutrophils can unexpectedly allow Streptococcus gordonii, a normally health-associated oral bacterium, to survive within the immune cells and to trigger the NLRP6 inflammasome, leading to increased IL-1{beta} release. In this study, we found that Porphyromonas gingivalis, a key periodontal pathogen, stimulates macrophages to allow S. gordonii survival and increased IL-1{beta} production. Using a mouse model of periodontal disease, we also found that without NLRP6 mice had less bone loss, less inflammation, and fewer neutrophils when P. gingivalis was present along with other oral bacteria. These results show that NLRP6 plays an important role in how oral bacteria work together to worsen periodontal disease.

Cryptococcus neoformans is the leading cause of fungal meningitis in immunocompromised patients. Alveolar macrophages are the first line of defense against Cryptococcus infection. Our previous study showed that deletion of Cdc50, the regulatory subunit of P4-ATPase (lipid flippase) complex, results in increased phagocytosis and macrophage killing, and avirulence in animal models. However, how fungal flippase dysfunction modulates Cryptococcus-macrophage interaction remains unknown. Here we identify Cdc50 as a central determinant of membrane lipid homeostasis, extracellular vesicle (EV) biogenesis and macrophage responses in C. neoformans. Our whole cell lipidomic analysis revealed that loss of Cdc50 disrupted membrane lipid homeostasis leading to phospholipid enrichment in cdc50{Delta} mutant, and a reduction in fatty acid production accompanied by pronounced ultrastructural defects in membrane architecture. Loss of Cdc50 also induced a hyper-vesiculating phenotype, with cdc50{Delta} producing significantly more extracellular vesicles (EVs) than wild type H99 cells. Lipidomic profiling of cdc50{Delta} EVs revealed enrichment of phospholipids, including phosphatidylserine (PS), indicating active lipid sorting during vesicle biogenesis. Functional analysis showed that EVs from the wildtype H99 suppress phagocytosis whereas cdc50{Delta} EVs enhance phagocytosis, indicating a differential macrophage priming. Despite increased PS externalization in cdc50{Delta} cells and EVs, macrophage recognition and uptake occur independent of PS-mediated efferocytosis pathways, including PS receptor MertK. Following macrophage uptake, cdc50{Delta} were intrinsically vulnerable to macrophage killing due to rapid phagosome acidification. Together, we demonstrate that Cdc50 dependent lipid homeostasis regulates EV production, lipid composition, membrane architecture and drives the intracellular fate of C. neoformans. IMPORTANCECryptococcus neoformans is the leading cause of fungal meningitis in immunocompromised individuals. Understanding how this pathogen evades host immune mediated clearance is essential for developing new treatment strategies. Here, we demonstrated that Cdc50, the regulatory subunit of fungal lipid flippase complex, regulates membrane lipid homeostasis that governs extracellular vesicles (EV) biogenesis and macrophage immune responses. Loss of Cdc50 drives global membrane lipid remodeling, hyper-production of phospholipid enriched EVs that enhance macrophage phagocytosis, while the wild-type EV reduce macrophage phagocytosis. Contrary to the prevailing assumption that phosphatidylserine (PS) externalization on the fungal surfaces mimics the mammalian "eat-me signal", we show fungal PS does not engage canonical PS receptor MertK, revealing a fundamental difference between fungal and mammalian PS biology. Furthermore, cdc50{Delta} cells are unable to resist phagosomal acidification, rendering them susceptible to macrophage killing. These findings establish how phospholipid homeostasis contributes to early host-pathogen interactions and serves as a compelling antifungal target in cryptococcosis.

Cryptococcus neoformans is an opportunistic fungus that causes pulmonary and central nervous system infections after entry into the lungs. We previously established that signaling through the adapter protein DAP12 inhibits the antifungal response of monocyte-derived macrophages and worsens the survival of mice after C. neoformans infection. However, the molecular mechanisms by which DAP12 signaling is initiated during cryptococcosis remain inadequately characterized. In this study, we identify triggering receptor expressed on myeloid cells 2 (TREM2) as a DAP12-associated receptor that is induced on murine monocytes and interstitial macrophages in the lungs in response to C. neoformans infection. TREM2 subsequently represses fungal uptake and M1 polarization by monocyte-derived macrophages. Using an in vitro binding assay, we find that both murine and human TREM2 can directly bind to C. neoformans and that the absence of the cryptococcal cell wall antigen {beta}-1,6-glucan disrupts these interactions. Overall, our findings suggest that the TREM2-DAP12 pathway plays an important inhibitory role in the host immune response to cryptococcal infection by impeding macrophage activation and phagocytosis of fungal cells. We also establish TREM2 as a receptor involved in direct fungal sensing of C. neoformans.

Neisseria gonorrhoeae (Ng) is the causative agent of gonorrhea, and the global spread of antimicrobial-resistant strains makes vaccine development a public health priority. Although messenger RNA (mRNA) vaccines have transformed protection against viral diseases, the platform remains in its infancy against pathogenic bacteria. Here, we evaluated the immunogenicity and protective efficacy of an mRNA-lipid nanoparticle (LNP) vaccine encoding the highly conserved outer membrane antigen BamA in the female mouse model of lower genital tract infection. We delivered BamA mRNA-LNPs via intramuscular (IM) or intranasal (IN) routes, with or without CpG ODN 2395, and measured antigen-specific antibody responses in serum and vaginal lavage samples. Both routes elicited robust BamA-specific antibodies that recognized diverse Ng isolates, including ceftriaxone-resistant strains. However, neither route accelerated bacterial clearance nor reduced bioburden, nor did either generate serum bactericidal activity. These findings show that BamA mRNA-LNPs are immunogenic but, as formulated, are not protective, and they pave the way for modifications to the construct, adjuvant, and route. To our knowledge, this is the first evaluation of an mRNA vaccine against Ng, establishing the platform as an amenable approach for gonococcal antigen testing. ImportanceNeisseria gonorrhoeae is a high-priority public health threat, and no licensed gonococcal vaccine exists. mRNA-lipid nanoparticle vaccines have transformed antiviral immunization but remain largely untested against bacterial pathogens. This study is the first to evaluate an mRNA vaccine against N. gonorrhoeae, using the conserved, essential outer membrane antigen BamA. We show that BamA mRNA-LNPs delivered by intramuscular or intranasal routes elicit robust, broadly cross-reactive antibodies that recognize diverse isolates, including ceftriaxone-resistant strains, even though the tested formulations did not confer protection in mice. By establishing mRNA-LNPs as a viable platform for gonococcal antigen testing, this work lays the foundation for optimizing constructs, adjuvants, and routes for developing mRNA-based vaccines against gonorrhea and other antimicrobial-resistant bacteria.

Small non-coding RNAs (sRNA) modulate diverse bacterial functions ranging from carbon metabolism to virulence gene expression. Previous research showed that the sRNA chaperone Hfq is critical for the fitness of Extraintestinal Pathogenic Escherichia coli (ExPEC), a major cause of both bloodstream and urinary tract infections (UTI). Using the reference ExPEC strain UTI89, we created deletion mutants to probe the effects of seven conserved Hfq-dependent sRNAs (DsrA, RprA, OxyS, RyhB, MicF, MicC, Spf) on resistance to oxidative stress. All of the sRNA mutants grew normally in replete lysogeny broth, but the spf and micC mutants exhibited additive effects upon challenge with reactive oxygen species generated by methyl viologen. In a murine UTI model, the spf mutant resembled the wild-type strain, whereas UTI89{Delta}micC was unable to effectively colonize the bladder despite behaving like wild type within the kidneys. This correlated with a greatly reduced ability of the micC mutant to survive within bladder epithelial cells and paralleled UTI89{Delta}micC defects in gut colonization, virulence in a sepsis model, and complement resistance. Although MicC downregulated expression of its only known target, OmpC, aberrant modulation of this porin did not entirely account for the decreased stress resistance of UTI89{Delta}micC. Rather, RNA-Seq, sRNA target predictions, and in vitro phenotypic assays revealed that MicC can impact multiple pathways linked to niche establishment, including motility, chemotaxis, and various metabolic processes. These data are consistent with MicC serving as a multifunctional regulator of ExPEC stress responses and niche-specific fitness through OmpC-dependent and - independent mechanisms. IMPORTANCEPathogenic strains of Escherichia coli are exceptionally common causes of diarrheal disease, urinary tract infection, sepsis, and meningitis. The ability of these pathogens to cause such a wide range of maladies is in part attributable to their ability to quickly adapt to and thrive within disparate and often hostile environments, including the gut, bladder, kidneys, and bloodstream. Adaptation to new environments requires rapid and precise changes in gene expression. To accomplish this feat, E. coli utilizes a suite of regulatory RNA called small RNA (sRNA). In this paper, we identified the sRNA MicC as a critical facilitator of E. coli fitness and virulence within diverse host environments via effects on the expression of multiple genes involved in bacterial motility, energy acquisition, and various other pathways. Delineating how sRNAs like MicC impact disease processes will aid the development of novel therapeutics to better combat E. coli infections.

Candidozyma auris (C. auris) is an emerging multidrug-resistant fungal pathogen, and its dissemination to the bloodstream and deep-seated organs is associated with high mortality. The limited antifungal armory and pipelines against C. auris pose a major challenge in disease management. Addressing this threat requires a deeper understanding of fungal virulence mechanisms that promote persistence and of host factors that drive susceptibility. Previous in vitro studies showed that iron enhances C. auris resistance to azoles and echinocandins, whereas iron chelation mitigates this effect. Here, we demonstrate that C. auris does not utilize cell-free heme or induce hemolysis but instead extracts and uses iron from transferrin to support growth and virulence. Deletion of the SIT1 siderophore transporter in C. auris attenuated fungal growth and reduced renal injury, while increased transferrin-iron saturation worsened disease outcomes in immunocompetent mice, highlighting the importance of transferrin-bound iron uptake. Mechanistically, C. auris exploits transferrin-bound iron to enhance ergosterol biosynthesis and activate antioxidant defenses, promoting resistance to neutrophil- and caspofungin-mediated killing. These findings identify elevated transferrin saturation as a novel host susceptibility risk factor for disseminated C. auris infection and reveal how iron availability reshapes fungal physiology to drive infection persistence.

Certain strains of the facultative intracellular bacterial pathogen Listeria monocytogenes are thought to invade cranial nerves in the gut and disseminate directly to the brainstem to cause rhombencephalitis in both humans and ruminants. Bacteria with actin tails were previously observed within neurons of naturally infected sheep, but the mechanism for how these neurotropic strains access the nervous system has not been well characterized. Using a foodborne mouse model of listeriosis, we show here that bypassing the gut phase of infection prevents colonization of the brain, confirming that invasion of the nervous system happens in the intestinal tract. L. monocytogenes did not efficiently invade neuronal cell lines, although they could replicate exponentially in the cytosol and form actin tails. Instead, the neurotropic strains displayed a preferential ability to invade enteric glial cells, a specialized subset of glia that support neurons and are critical for intestinal homeostasis. Using an in vitro co-culture system, we demonstrated that neurotropic L. monocytogenes could readily invade enteric glial cells and use ActA-mediated actin-based motility to spread to adjacent neurons. These results suggest that invasion of enteric glial cells is a novel virulence strategy that can promote brainstem infection following foodborne transmission of L. monocytogenes. IMPORTANCEThis study provides further evidence for dissemination of neurotropic strains of L. monocytogenes from the gut directly to the brain via axonal migration using foodborne mouse model of listeriosis. It is the first report showing that enteric glial cells, a specialized subset of cells in the gut that support intestinal neurons, are susceptible to pathogenic bacterial infection.

Trichomonas vaginalis is the leading cause of non-viral sexually transmitted infections and it is associated with comorbidities that affect female health. Lactobacillus iners is one of the most predominant bacteria in the cervicovaginal microbiome. As so, both microbes are likely to encounter one another upon T. vaginalis infection. To our knowledge, the interaction of both microbes has not been previously investigated. Here, we report that T. vaginalis and L. iners bind to one another at early time points of co-incubation. Using imaging flow cytometry and scanning electron microscopy, we capture the dynamics of this microbe-microbe association. We observed active remodeling of the T. vaginalis cell surface leading to thin-membrane protrusions that make contact with L. iners. Larger T. vaginalis membrane extensions that surround and engulf L. iners were also visible. These T. vaginalis-L. iners interactions ultimately lead to a reduction of L. iners viability while T. vaginalis viability was unaffected by exposure to L. iners. Inhibition of actin polymerization blocked T. vaginalis antibacterial activity against L. iners. Together our findings reveal novel insight about T. vaginalis-L. iners interactions and highlight a new T. vaginalis pathogenic effect.

Bacteria routinely exhibit unexpected phenotypic and molecular changes in response to spaceflight and spaceflight-analogue conditions, yet the mechanisms by which they sense and respond to these low fluid shear environments are not fully elucidated. We previously demonstrated that spaceflight and low shear modeled microgravity (LSMMG) altered motility and chemotaxis gene expression in Salmonella enterica serovar Typhimurium (S. Typhimurium), raising the possibility that flagella mediate responses of the pathogen to these environments. Herein, we investigated whether LSMMG culture alters S. Typhimurium motility and examined the role of flagella in regulating pathogenesis-associated stress and infection phenotypes. LSMMG enhanced the swimming motility of wild-type S. Typhimurium relative to 1xg controls; a trend which persisted even in the absence of the global stress response regulators Hfq and RpoS. This finding was unexpected, as {Delta}hfq mutants are typically defective for motility under conventional culture conditions. Motility was also observed in the flagella-deficient {Delta}flhDC mutant following LSMMG and 1xg culture, although the relative motility pattern differed relative to wild-type. Collectively, these results indicate that flagella contribute to LSMMG-enhanced motility, but are not strictly required under these conditions. Conditioned supernatant exchange demonstrated that LSMMG-induced motility changes are cell-intrinsic rather than mediated by extracellular factors. While flagella were dispensable for many pathogenesis-related phenotypes tested, their deletion selectively altered the magnitude of LSMMG-associated thermal stress and intracellular survival in human intestinal epithelial cells. Together, these findings demonstrate that motility and pathogenesis-related responses in S. Typhimurium are governed by multiple regulatory pathways that differentially respond to LSMMG and 1xg conditions. IMPORTANCESpaceflight and spaceflight-analogue conditions alter bacterial physiology in unexpected ways that are important for pathogenesis, yet the mechanisms by which bacteria sense and respond to low fluid shear environments remain incompletely understood. This study shows that low shear modeled microgravity (LSMMG) enhances Salmonella Typhimurium motility and produces unexpected motility phenotypes in mutants lacking Hfq or the flagellar master regulator FlhDC. These findings indicate that flagellar biosynthesis contributes to LSMMG-enhanced motility but is not strictly required for motility under these conditions. We also suggest that flagella influence the magnitude of selected stress and infection phenotypes rather than serving as an absolute requirement for LSMMG responsiveness. Together, these results highlight the complexity of bacterial mechanotransduction under simulated microgravity conditions and advances our understanding of how a foodborne pathogen adapts to physiological low fluid shear environments encountered both in space and during terrestrial infection of the intestinal tract.

Cathelicidins are a class of antimicrobial peptides (AMPs) that are part of the first line of defense of the innate immune system. While cathelicidin-derived peptides such as LL-37 can be directly bactericidal, Streptococcus pyogenes (Spy; Group A Streptococcus) is highly resistant to killing. Furthermore, Spy detects LL-37 through the CovRS two-component system to regulate its virulence factors. One effect of this signaling is the repression of expression of the bacterial protease SpeB. Prior work has also shown that SpeB, along with other bacterial proteases can cleave LL-37. However, it is unclear if SpeB cleavage of LL-37 impacts antimicrobial function and CovRS signaling activity. Using a genetic approach, we show that the presence SpeB did not significantly impact the killing of Spy by LL-37 relative to other known resistance factors. Furthermore, while SpeB cleaves LL-37, CovRS maintains sensitivity to LL-37 fragments. These results indicate that SpeB cleavage of LL-37 does not negatively impact virulence factor regulation in Spy.

To establish infection and cause disease, the intracellular pathogen Shigella must successfully navigate a series of host defenses and distinct microenvironments within the human body. One way Shigella navigates these enviroments is by using the secondary messenger c-di-GMP, which regulates many different bacterial behaviours. C-di-GMP is synthesized by diguanylate cyclases (DGCs) and broken down by c-di-GMP specific phosphodiesterases (PDEs). In this study, we investigated how Shigellas c-di-GMP specific PDEs impact c-di-GMP turn-over and subsequently biofilm and virulence phenotypes. We knocked out each of Shigellas six c-di-GMP specific PDEs to determine how these PDEs impact biofilm, virulence and c-di-GMP levels within the bacterial cell. We found that these PDEs negatively regulate c-di-GMP levels while modulating Shigellas virulence and biofilm behaviour. We also noted that altering expression of these Shigella PDEs changes bacterial cell size. Transcriptome analysis revealed that a Shigella {Delta}pdeB strain showed reduced expression of many genes, including the virulence genes ipgD and ipgE, as well as genes associated with lipid metabolism. We confirmed that a Shigella {Delta}pdeB strain had altered levels of stearic acid, and expression of pdeB alters Shigella antibiotic susceptibility. This study highlights the complexities of c-di-GMP signaling in regulating numerous Shigella pathways.

Streptococcus pneumoniae asymptomatically colonizes the nasopharynx but can invade the lower respiratory tract to cause life-threatening disease, particularly in older adults. However, whether the initial site of bacterial deposition following intranasal inoculation determines disease progression has not been directly examined. Here, we developed a near-infrared (NIR) fluorescence imaging approach using indocyanine green (ICG)-labeled S. pneumoniae TIGR4 to visualize early bacterial distribution in real time. ICG labeling by simple mixing, without genetic or chemical modification, neither impaired bacterial growth at 33 or 37{degrees}C, nor altered acid tolerance. Continuous video imaging during the first 10 min of infection resolved two distinct patterns: bacteria confined to the nasopharynx (colonization) and those aspirated into the lower respiratory tract (aspiration). Kaplan-Meier analysis revealed markedly higher mortality in the aspiration group in both young (hazard ratio = 7.9) and aged (hazard ratio = 8.4) mice, despite a 10-fold lower inoculum used for aged animals, with deaths beginning on day 3. Systemic profiling of blood at 24 h by RNA sequencing and plasma proteomics revealed that early aspiration in aged mice was associated with the activation of inflammatory and hematopoietic programs, enrichment of complement and coagulation cascades, and phagocytic pathways. Together, these findings establish aspiration into the lower respiratory tract as a trigger of severe pneumococcal disease and introduce real-time NIR imaging as a technique for linking early infection dynamics to systemic host responses.

A low abundance or absence of protective lactobacilli during acute trichomoniasis is a well-known phenomenon that was reported in multiple studies and is the hallmark of a T. vaginalis (TV) infection. However, a crucial question that remains unanswered is whether alterations in the lactobacilli population precede TV infection or whether the parasite plays an active role in lactobacilli disappearance. Our findings showed that TV efficiently phagocytosed the dominant Lactobacillus species L. jensenii (LJ). Phagocytosis proceeds via a pseudopodia-independent mechanism reminiscent of sinking with a preference for viable cells. The presence of viable LJ leads to an increase in secretion of 27 TV proteins, including TvGH25 lysozyme. This enzyme cleaves peptidoglycan, a major component of the bacterial cell wall. TV overexpressing TvGH25 effectively lowers the bacterial cell count, evidencing the enzyme's antimicrobial potential. These data support the notion that TV cells can suppress the Lactobacillus population through a combination of targeted secretory response and phagocytic activity, revealing novel potential targets for developing alternative therapeutic strategies against trichomoniasis.

S. mutans and V. parvula cooperate in dental plaque to assemble a healthy biofilm and are associated with increased caries risk. S. mutans produces lactic acid from carbohydrates resulting in a final biofilm pH[~]4, while V. parvula metabolizes lactate to acetic and propionic acids resulting in pH[~]5. This process results in healthier biofilms that still generate a pH capable of demineralizing tooth surfaces (pH<5.5). The purpose of this study was to identify V. parvula genes required for deacidification of S. mutans biofilms and determine whether the ability of V. parvula to deacidify S. mutans biofilms correlates with enhanced biofilm health. Using transposon mutagenesis in V. parvula we identified several genes required for deacidification of S. mutans biofilms. These included numerous V. parvula transposon mutations in the previously unstudied lutABC lactate utilization operon. To assess biofilm health, S. mutans in the presence of various V. parvula mutants were stained with a LIVE/DEAD stain and imaged by fluorescence microscopy. An intact lutABC operon was required to enhance biofilm health, as demonstrated by plasmid-based complementation of a lutB transposon mutant. Transposon insertions in other loci unrelated to deacidification had no impact on biofilm health. Addition of HEPES buffer at the time of S. mutans biofilm assembly prevented full acidification of the biofilm and resulted in improved biofilm health, even without the addition of V. parvula. Finally, we found V. parvula can use either nitrate or fumarate as a final ETC electron acceptor during lactate utilization. In all, we found the lutABC lactate utilization operon of V. parvula is critical for the ability of V. parvula to deacidify S. mutans biofilms and promote biofilm health. Interfering with this pathway would interrupt the mutually beneficial relationship between S. mutans and V. parvula that leads to their co-association in caries, root caries, and early childhood caries.