Enteric viruses are major causes of gastrointestinal disease worldwide and are primarily transmitted through the fecal-oral route. Upon entering the gastrointestinal tract, these viruses encounter the dense and diverse population of intestinal microbiota, leading to frequent interactions with commensal bacteria. Over the past decade, numerous studies have demonstrated that bacteria can enhance enteric viral replication, stability, and overall pathogenesis. Although direct binding interactions between enteric viruses and bacteria have been observed, the molecular determinants governing these interactions remain poorly understood. Defining the viral and bacterial factors that mediate binding is, therefore, critical for understanding how bacteria can influence viral infection, transmission, and disease. Here, in this minireview, we summarize the current findings on enteric viral-bacterial interactions, with a focus on the mechanisms of viral binding to bacteria and the implications of these interactions on enteric virus pathogenesis and transmission.
Interferons (IFNs) coordinate host defense at the lung barrier by linking pathogen recognition to epithelial and immune-cell responses. Their effects are highly context-dependent and can be protective or pathological according to IFN class and ligand, cellular source and target, and the timing and duration of signaling. In this minireview, we compare the induction of type I, II, and III IFNs during respiratory viral infection and pulmonary Aspergillus fumigatus infection, highlighting epithelial-led antiviral sensing and predominantly myeloid-centered antifungal sensing. We then examine how receptor distribution, ligand identity, JAK-STAT complex assembly, and integration with inflammatory pathways generate cell type-specific transcriptional and functional programs. Finally, we discuss three interconnected consequences of IFN crosstalk at the respiratory barrier: regulation of epithelial permeability and repair, licensing or pathological reprogramming of immune effector cells during primary infection, and loss of coordination during viral-fungal coinfection, including influenza-associated and COVID-19-associated pulmonary aspergillosis. Collectively, the available evidence supports a model in which spatially restricted and appropriately timed IFN responses promote pathogen control and preserve barrier function, whereas excessive, prolonged, or mistimed signaling impairs epithelial recovery, disrupts phagocyte activity, and increases susceptibility to secondary fungal invasion. Defining these context-dependent circuits may guide therapeutic strategies that modulate IFN signaling with greater temporal and cell type specificity.
Providencia alcalifaciens is a gut commensal bacterium and also an emerging enteric pathogen associated with sporadic infections and outbreak cases in humans. The most notable outbreak caused by this bacterium occurred in 1996 in Fukui Prefecture, Japan, affecting 270 individuals. However, the pathogenic mechanisms responsible for this outbreak remain unknown. In this study, we identified the key virulence determinants of the Fukui outbreak strains through genomic and functional analyses. These strains uniquely carry a ~162 kb large plasmid encoding a type III secretion system (T3SS) closely homologous to the Salmonella SPI-1 T3SS. We also show that the plasmid-encoded T3SS (T3SSp) constitutes a functional secretion system and is essential for the pathogenicity of the Fukui outbreak strain, including the invasion of cultured epithelial cells and the induction of diarrhea in a rabbit model. Secretome analysis identified effectors secreted in a T3SSp-dependent manner, among which PipA-sharing limited sequence similarity with SipA, a SPI-1 T3SS effector-plays a crucial role in inducing diarrhea. Ectopic expression of PipA in HeLa cells caused focal accumulation of F-actin, indicating its cytoskeleton-modulating activity. Comparative genomics with other Providencia species revealed the dissemination of the large plasmid, with structural variations among enteropathogenic strains of P. alcalifaciens and Providencia rustigianii associated with clinical cases in humans and animals. Thus, our findings underscore the molecular basis of P. alcalifaciens pathogenicity in the Fukui outbreak and highlight the significance of the large plasmids encoding T3SS in driving pathogenic evolution among Providencia species.
The high virulence and pathogenesis of Francisella tularensis (Ft), responsible for tularemia, made it a target for bioweapon development during the "Cold War." This prompted investigation of vaccines, starting with an older vaccine candidate denoted Ft live vaccine strain (LVS). LVS has limited efficacy against aerosol challenge with Ft subsp. Tularensis (Type A), the most virulent subtype. However, no vaccines, including LVS, have been licensed in the U.S. Animal studies indicate that Ft vaccines induce protective T-cell-mediated immune responses. In previous studies, analyses of leukocytes from vaccinated animals that were restimulated in vitro facilitated screening and selection of vaccines for more extensive in vivo evaluation. These studies demonstrated that a novel live attenuated vaccine strain derived from Type A Ft, denoted ΔclpB, was protective against aerosol challenge in animal models. In this study, we focused on evaluating immune responses immediately after vaccination of Fischer 344 rats with LVS and ΔclpB using multiple analytical approaches. While humoral immune responses were comparable between the two vaccines, we identified multiple differences in immune responses by analyses of peripheral blood leukocytes by flow cytometry and by relative gene expression. In particular, vaccination with LVS or ΔclpB resulted in differential expression of multiple genes at selected time points. Gene expression, in turn, predicted activation or inhibition of multiple biological pathways. These results suggest that immune responses shortly after primary vaccination may discriminate vaccines with different degrees of in vivo protection that could potentially be used as correlates of protection and extrapolate efficacy from animals to people.
Pseudomonas aeruginosa is a common opportunistic pathogen that causes chronic lung infections in individuals with cystic fibrosis. Despite advances in therapies that restore cystic fibrosis transmembrane conductance regulator function, persistent colonization of the airway remains a major clinical challenge. Reduced clearance of P. aeruginosa from the cystic fibrosis airway has been associated with the increased activity of histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase that decreases microtubule acetylation and stability. In this study, we investigated the role of HDAC6 in modulating interactions between P. aeruginosa and cystic fibrosis airway epithelial cells. Pharmacologic inhibition of HDAC6 significantly reduced bacterial adherence in both mouse and human cystic fibrosis epithelial cells. Genetic deletion of HDAC6 produced similar effects, while knockout of a microtubule-stabilizing protein increased bacterial adherence, mimicking the cystic fibrosis phenotype. HDAC6 inhibition also reduced bacterial internalization, although to a lesser extent compared to adherence. These results suggest that microtubule destabilization contributes to the enhanced colonization of cystic fibrosis airways by P. aeruginosa. Targeting host microtubule regulatory pathways, particularly by inhibiting HDAC6, may represent a promising host-directed strategy to limit early bacterial attachment and reduce the risk of chronic infection in cystic fibrosis.
ABSTRACT Burkholderia pseudomallei causes the tropical disease melioidosis. Host mortality primarily results from sepsis-related complications and associated cytokine release. Lipopolysaccharide (LPS), a major virulence factor and mediator of sepsis, is modified by B. pseudomallei in response to external stimuli. LpxO and PagL are lipid A-modifying proteins encoded by the B. pseudomallei genome to decrease recognition by host defenses. The contribution of lpxO and pagL-dependent lipid A modifications to outer membrane permeability and host response has not been clearly demonstrated. Mono-phosphorylated lipid A (MPLA) is known to maintain LPS-specific immunogenicity while reducing endotoxicity and has been used extensively as a vaccine adjuvant. Generation of MPLA can be mediated through LpxE. In this work, a panel of defined lipid A modification strains in B. pseudomallei was generated to study effects on bacterial physiology and cytokine expression in macrophages. Knockout of lpxO and pagL resulted in dehydroxylation and penta-acylation of lipid A, respectively, and reduced membrane permeability of B. pseudomallei. Expression of lpxE removed one phosphate group as measured by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and caused increased membrane permeability. Outer membrane vesicles (OMVs) from the generated mutants were used to study the immunological effect of lipid A modifications on human monocyte-derived macrophages. Macrophages treated with OMVs isolated from lpxE-expressing strains showed significant increases in gene expression of proinflammatory cytokines tumor necrosis factor and interferon gamma compared to wild-type B. pseudomallei OMVs. These results show the importance of lipid A modifiers LpxO and PagL for B. pseudomallei physiology and highlight the immunostimulatory effect of lpxE-modified OMVs that could enhance OMV vaccine technology targeting melioidosis.
Pasteurella multocida causes several severe animal diseases, including hemorrhagic septicemia in ungulates. Strains of P. multocida are differentiated into five capsular and nine lipopolysaccharide genotypes. To date, only P. multocida strains that produce capsule types B and E have been recovered from cases of hemorrhagic septicemia. The structure of the type B capsule has recently been determined to be a repeating main chain of N-acetyl glucosamine (GlcNAc) and N-acetyl mannosaminuronic acid (ManNAcA), with a fructose and glycine side chain. In this study, we investigated the role of proteins involved in capsule biosynthesis in P. multocida strain M1404, a bison hemorrhagic septicemia isolate that produces a type B capsule. Using capsule absorbance assays, Alcian blue stains, and capsule Western blots to assess capsule production and export, as well as in-source collision-induced dissociation mass spectrometry to determine capsule structure, we show that BcbC is likely the capsule synthase; BcbE and BcbG are involved in fructose side chain addition, and BcbH is likely the glycine transferase. Furthermore, we show that inactivation of bcbE, bcbG, or bcbH significantly decreases overall capsule production in P. multocida strain M1404. Additionally, heterologous expression of bcbABCDI in a P. multocida strain VP161 (type A) capsule mutant allowed production of a repeating GlcNAc-ManNAcA monomer in this strain background that was indistinguishable from the type B main chain, indicating that these genes are necessary and sufficient for synthesis of the main repeating type B polysaccharide.
Macrolide antibiotics are commonly prescribed to treat Haemophilus influenzae respiratory tract infections. Studies have primarily focused on emerging H. influenzae strains with acquired macrolide resistance, while the bacterium's intrinsic resistance to antibiotics has been underexamined. Here, we used a genome-wide approach of transposon insertion-site sequencing to screen an H. influenzae mutant library grown in sub-inhibitory doses of the macrolide antibiotic clarithromycin (CLR) to identify 33 genes involved in intrinsic CLR resistance. Almost half of these genes are also needed for survival in the mouse lung. We focused on candidate genes necessary for both intrinsic macrolide resistance and lung survival. Two of these genes affect the outer-membrane composition of H. influenzae, orfH and omp26. Deletions of these genes in Rd and nontypeable H. influenzae clinical isolates, Hi375 and NT127, conferred sensitivity to CLR and polymyxin B and increased membrane permeability to ethidium bromide (EtBr). The omp26 mutant was sensitive to killing by human serum. Deletions of orfH or omp26 in an acrR mutant strain overexpressing a multidrug efflux pump abrogated resistance of the acrR mutant to CLR and restored permeability to EtBr. Thus, deletion of these genes not only mitigates the effects of an acquired resistance mechanism but also remarkably overrides it. Complementation of these deletion mutations restored CLR resistance and decreased permeability to EtBr. Our results indicate that the subset of genes with dual roles in intrinsic resistance and host lung survival may provide potential novel combination antimicrobial therapeutic targets.
Hospital-acquired infections (HAIs) represent a major global health burden, leading to increased patient morbidity, mortality, and healthcare costs. These infections frequently occur following patient contact with microbial pathogens persisting on indwelling devices and other abiotic surfaces in the hospital environment. A key driver of this persistence is the ability to survive the total loss of water, a phenomenon known as desiccation tolerance. While desiccation tolerance has been studied across various kingdoms of life, comparatively little is known about the molecular mechanisms that enable bacterial pathogens to tolerate extreme water loss. In this review, we will examine the consequences of desiccation stress in bacteria and highlight conserved mechanisms by which bacterial cells tolerate drying, including spore formation, capsule synthesis, osmolyte accumulation, expression of intrinsically disordered proteins, and other protective mechanisms. We will also examine the mechanisms and consequences of desiccation tolerance in several clinically significant nosocomial pathogens and review emerging evidence that desiccation stress responses may influence other bacterial traits, such as antimicrobial resistance, polymicrobial community interactions, and virulence. Understanding the mechanisms by which nosocomial pathogens tolerate desiccation may reveal novel strategies to disrupt pathogen reservoirs and reduce future HAI transmission.
Pseudomonas aeruginosa (Pa) is a ubiquitous, opportunistic nosocomial pathogen that poses a significant threat due to its innate and acquired multidrug resistance. Novel vaccine strategies are urgently needed for vulnerable populations, many of which harbor pre-existing immunity from prior encounters with Pa. Here, we evaluated a multivalent subunit vaccine combining type III secretion system (T3SS) antigens and exolysin A (ExlA) in a nanoemulsion formulation using a clinically relevant murine pulmonary pre-exposure model. This approach allowed us to determine whether vaccination could overcome the limitations of the host's initial, ineffective immune response. Vaccination fundamentally transforms suboptimal baseline memory into a potent, multi-faceted Th1/Th17-polarized response. This globally transformed signature was characterized by significantly enhanced antigen-specific IFN-γ and IL-17A production, both locally and systematically in the lung, with exceptionally large biological effect sizes (Cohen's d values reaching 21.35). By utilizing log10 transformation to accurately reflect pathogen growth kinetics, we demonstrated that this vaccine-augmented immunity conferred statistically significant protection following heterologous challenge. In the twice-exposed cohort, vaccination promoted superior bacterial clearance of the T3SS-positive strain, though clearance of the ExlA-positive strain was not enhanced, potentially due to immunological interference from pre-existing T3SS memory. In the thrice-exposed cohort, a functional protective threshold was observed, where high levels of natural immunity matched the vaccine-induced clearance levels. Our findings established that our vaccine formulation can effectively boost and redirect pre-existing immunity, offering a promising approach to overcome the limitations of natural exposure and protect at-risk individuals from diverse Pa infections.
The pathogenic yeast Cryptococcus neoformans causes life-threatening meningoencephalitis in individuals with compromised immune systems. The ability of the fungus to cause disease depends on key cell-surface features, such as a polysaccharide capsule that protects it from the mammalian immune system. However, the mechanisms by which C. neoformans traffics polysaccharide capsule, melanin, and other materials to the cell surface are poorly understood. In this study, we employed mutants lacking specific subunits of the adaptor protein complex 1 (AP-1) to investigate its role in the elaboration of virulence-related materials at the cell surface. Importantly, the mutants displayed multiple defects, including alterations in capsule size and cell morphology, and defects in melanin production and urease secretion. Together, these results support the key observation that the AP-1 complex is required for C. neoformans survival in phagocytic cells. Together, our findings provide insights into the endomembrane trafficking machinery required for fungal pathogenesis.
Cryptococcal meningoencephalitis is among the most prevalent invasive fungal diseases, posing a threat to immunocompromised individuals and representing a growing global health concern. The mechanisms of cryptococcal trafficking of virulence factors during disease are incompletely understood. Adaptor protein (AP) complexes play crucial roles in intracellular trafficking by orchestrating the sorting of macromolecular cargo and serving as essential components of the endocytic and secretory pathways. In a recent study, we demonstrated that Cryptococcus neoformans cells lacking the AP-1 complex subunits exhibit impaired elaboration of virulence factors and fail to survive in the harsh conditions of the macrophage phagolysosome. Although we characterized the phenotypes of AP-1 deficient cells in vitro, the contribution of this complex to pathogenesis remains unexplored. In this study, we show that mutants deficient in AP-1 complex subunits are either avirulent or exhibit attenuated virulence in a murine inhalational model. Loss of the small subunit resulted in the formation of granuloma-like lesions in mouse lungs with early containment of infection but eventual mortality, whereas mutants lacking the large subunits were rapidly cleared by mice. The delayed onset of disease in mice caused by mutants lacking the small subunit was marked by delayed weight loss and increased respiration rate, yet the mutant exhibited enhanced dissemination during late-stage infection, coinciding with waning immune responses and elevated collagen deposition. These findings demonstrate that deficiencies in the AP-1 complex impair C. neoformans virulence, reveal distinct roles for individual subunits, and identify the complex as a potential target for therapeutic intervention in cryptococcosis.
Innate immune signaling plays a key role in host response to infection, yet the pattern recognition receptors that detect non-model gut-associated yeasts remain poorly defined. Here, we investigated macrophage sensing of Debaryomyces hansenii, a food-derived yeast that we found to be enriched within intestinal ulcers of Crohn disease (CD) patients. Using a cell surface receptor antibody screen of bone marrow-derived macrophages infected with a CD patient isolate of D. hansenii, we showed that D. hansenii-induced macrophage activation characterized by increased expression of co-stimulatory molecules, MHC-II, and pattern recognition receptors, including the C-type lectin receptor Dectin-1. Antibody blockade experiments showed both Dectin-1 and complement receptor 3 subunit CD11b were required for phagocytosis of D. hansenii, while Dectin-1 was uniquely required for production of the pro-inflammatory cytokine tumor necrosis factor (Tnf). CRISPR-Cas9-mediated deletion of Dectin-1 phenocopied antibody neutralization effects on phagocytosis. Furthermore, deletion of Dectin-1 or its downstream signaling adaptor molecule Card9 resulted in reduced Tnf secretion in response to D. hansenii. Dectin-1-mediated uptake of D. hansenii was observed in primary bone marrow-derived macrophage and dendritic cells, as well as across the spectrum of macrophage polarization states. Together, these findings define the role of Dectin-1-Card9 signaling axis in innate immune cell sensing of D. hansenii. These findings support the emerging relevance of innate immune recognition of a yeast in Crohn disease pathogenesis.
A consequence of the Borrelia (Borreliella) burgdorferi lifecycle is that a "standard" host-pathogen interaction involving the spirochete is challenging to define. The bacteria continuously cycle between vertebrate and invertebrate hosts, each inflicting substantially different selective pressures on the spirochete. Focusing solely on vertebrate infections, the spirochete can be found in numerous hosts in nature, including deermice, shrews, birds, and lizards. The immune pressures that the bacteria face in each of these hosts are variable and are thought to drive broad intraspecific variation across B. burgdorferi strains-with different strains appearing to be avian adapted, mammalian adapted, or generalist. In addition to its natural zoonotic hosts, B. burgdorferi can infect humans and cause Lyme disease. Here, natural human genetic variation can drive dramatically different disease outcomes, as some individuals seem capable of killing the bacteria before it can successfully colonize, while others are predisposed to continue reacting to the pathogen even long after it is killed by antibiotics. In this review, we describe studies investigating how host and bacterial diversity affect B. burgdorferi infection outcomes, with an emphasis on how this variability impacts the bacteria's spread in nature and Lyme disease severity during human infection.
Recently approved malaria pre-erythrocytic vaccines are important public health tools for the reduction of malaria morbidity and mortality. However, these vaccines have shown reduced protection in some of the most vulnerable age groups, require multiple dosing, and have the potential for parasite escape. These limitations have motivated the development of vaccines targeting multiple stages of the parasite life cycle. To ensure a robust pipeline, pre-clinical evaluation of additional malaria vaccine candidate antigens remains a key priority. The P. falciparum cysteine protease inhibitor falstatin can limit the cysteine protease activity of falcipains, controlling proteolysis at the site of merozoite entry on erythrocytes, thereby facilitating parasite invasion. Antibodies against falstatin have been shown to reduce the ability of merozoites to invade erythrocytes, raising the possibility that naturally acquired antibody responses could confer a similar advantage to the host. Here, IgG responses to full-length falstatin (falstatinFL) and two smaller, conserved regions (falstatin35-68 and falstatin289-335) were determined at the pre-malaria season baseline in a prospective cohort study conducted in Kalifabougou, Mali. The presence of anti-falstatin IgG was not associated with reduced risk of clinical malaria overall. However, baseline falstatin seropositivity predicted a non-statistically significant reduction in risk of febrile malaria within the first 30 days after incident parasitemia (log-rank statistic 3.2, P = 0.072). Using in vitro growth inhibitory assays, neither affinity-purified anti-falstatin289-335 IgG nor affinity-purified anti-falstatinFL IgG consistently inhibited P. falciparum blood-stage parasites. Larger cohort studies would be needed to confirm whether pre-existing anti-falstatin antibodies can delay the progression of malaria symptoms after blood-stage infection.
Clostridioides difficile infection (CDI) causes the majority of identifiable antibiotic-associated diarrhea. Epidemiological studies have shown that biological human females are more susceptible to CDI than males. In this study, we show that female mice developed more severe CDI than males under all conditions tested. We found time-delayed effects of the female estrus cycle on CDI. Indeed, animals in proestrus at any time during CDI progression developed severe signs 1-2 days later. In contrast, animals that were in the estrus stage were protected. Consistent with the delayed effect of the estrous cycle on CDI, we found that pre-infection levels of the sexual hormone prolactin (PRL), immunoglobulin IgG2b, cytokine IL-1β, cytokine G-CSF, and chemokine KC (CXCL1) were the primary nodes of a complex network that correlated with CDI symptomatology on the day after spore challenge. Similarly, we found that the pre-infection levels of sex hormone progesterone, sex hormone luteinizing hormone (LH), immunoglobulin IgG1, chemokine eotaxin, and chemokine IP-10 (CXCL10) were the primary network nodes that affected CDI severity, but with a 2-day delay. As expected, early post-infection levels of immunoglobulins, cytokines, and chemokines formed a hormone-independent network that concurrently correlated with CDI severity. Interestingly, early post-infection levels of FSH, together with cytokine IL-1β and chemokine KC (CXL1), were the main nodes of a network that affected CDI 2 days later, during the recovery phase of the infection. In summary, we show that murine female sexual hormones affect CDI progression, probably by affecting the immune system both before infection and during disease development.
ABSTRACT Macrophage antimicrobial programs are regulated not only by transcriptional networks but also by RNA processing mechanisms affecting signal transduction and effector responses. One such mechanism, alternative polyadenylation (APA), determines mRNA fate by changing the length of the 3′ untranslated region (3′ UTR). However, our understanding of the impact of APA on antibacterial functions and how we can manipulate it to influence infection outcomes remains limited. In this study, we identify the APA regulator CFIm25 (NUDT21) as a promoter of macrophage defense against Salmonella enterica serovar Typhimurium (STM). STM infection drives macrophages toward an M2-like immunosuppressive state conducive to bacterial survival, and we show that CFIm25 levels are concurrently reduced during this transition. Overexpression of CFIm25 in infected macrophages blocks STM-induced 3′ UTR lengthening of the key immune regulators TAB2 and TBL1XR1, restoring their mRNA and protein expression. Sustained CFIm25 activity reduces intracellular bacterial burden, enhances macrophage survival, increases reactive oxygen species and nitric oxide production, suppresses arginase activity, and preserves M1-associated surface marker expression and pro-inflammatory cytokine secretion. Mechanistically, TAB2 and TBL1XR1 knockdown studies demonstrate that the antibacterial effects of CFIm25 depend on these CFIm25 targets, which are important for activating MAPK and NF-κB signaling pathways. Furthermore, CFIm25 depletion increased the recovery of intracellular STM from the infected cells. Together, these findings identify APA regulation as a potential target for boosting innate immune defenses against chronic bacterial infections.
ABSTRACT Pseudomonas aeruginosa produces multiple toxins and exoenzymes that contribute to its survival and ability to cause disease. In the current study, we examined whether the type II-secreted cytotoxin Exotoxin A (ToxA) is required for P. aeruginosa growth and disease severity in infected murine corneas. Using Δ toxA mutants and complemented strains on a PAO1 background, we report that although ToxA is produced during corneal infection, ToxA deletion did not significantly affect bacterial replication, neutrophil recruitment, or disease severity in infected corneas. These findings contrast with an earlier study identifying a role for ToxA in P. aeruginosa keratitis.
ABSTRACT Competition between organisms is a ubiquitous feature of life on Earth and a major driver of evolutionary innovation. As Earth’s most diverse and abundant organisms, bacteria employ numerous strategies to inhibit the growth of competitors, ranging from the production of diffusible antibiotic metabolites to the secretion of sophisticated protein toxins. Although secreted antibacterial protein toxins have been extensively studied in gram-negative bacteria, analogous systems employed by gram-positive organisms remain comparatively poorly understood. Recent work has shed light on a widespread family of secreted protein toxins associated with co-secreted serine proteases that likely mediate interbacterial competition among gram-positive species. These systems, termed a nti b acterial p rotein (ABP) systems, consist of a secreted polymorphic t oxin (AbpT), a co-secreted serine p rotease (AbpP), and a cytoplasmic i mmunity protein (AbpI). Following proteolytic processing, ABP toxins inhibit the growth of a remarkably diverse range of gram-positive bacteria spanning the phyla Bacillota and Actinomycetota. Existing evidence suggests that these proteins combine the properties of cationic antimicrobial peptides with those of classical polymorphic antibacterial toxins, potentially enabling toxin delivery into distantly related bacteria. In this review, we summarize the current understanding of the organization, mechanisms, and ecology of ABP systems; discuss major outstanding questions regarding toxin import and native biological function; and highlight opportunities for future mechanistic and biotechnological investigation in this emerging field.
