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Spore formation is essential for the bacterial pathogen and obligate anaerobe, Clostridioides (Clostridium) difficile, to transmit disease. Completion of this process depends on the mother cell engulfing the developing forespore, but little is known about how engulfment occurs in C. difficile. In Bacillus subtilis, engulfment is mediated by a peptidoglycan degradation complex consisting of SpoIID, SpoIIP and SpoIIM, which are all individually required for spore formation. Using genetic analyses, we determined the functions of these engulfment-related proteins along with the putative endopeptidase, SpoIIQ, during C. difficile sporulation. While SpoIID, SpoIIP and SpoIIQ were critical for engulfment, loss of SpoIIM minimally impacted C. difficile spore formation. Interestingly, a small percentage of ∆spoIID and ∆spoIIQ cells generated heat-resistant spores through the actions of SpoIIQ and SpoIID, respectively. Loss of SpoIID and SpoIIQ also led to unique morphological phenotypes: asymmetric engulfment and forespore distortions, respectively. Catalytic mutant complementation analyses revealed that these phenotypes depend on the enzymatic activities of SpoIIP and SpoIID, respectively. Lastly, engulfment mutants mislocalized polymerized coat even though the basement layer coat proteins, SpoIVA and SipL, remained associated with the forespore. Collectively, these findings advance our understanding of several stages during infectious C. difficile spore assembly.

The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile , cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis , DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB , which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes . IMPORTANCE Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid (DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable for C. difficile spore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms.

Sporulation allows bacteria to survive adverse conditions and is essential to the lifecycle of some obligate anaerobes. In Bacillus subtilis, the sporulation-specific sigma factors, σ(F), σ(E), σ(G), and σ(K), activate compartment-specific transcriptional programs that drive sporulation through its morphological stages. The regulation of these sigma factors was predicted to be conserved across the Firmicutes, since the regulatory proteins controlling their activation are largely conserved. However, recent studies in (Pepto)Clostridium difficile, Clostridium acetobutylicum, Clostridium perfringens, and Clostridium botulinum have revealed striking differences in the order, activation, and function of sporulation sigma factors. These studies indicate that gene conservation does not necessarily predict gene function and that new mechanisms for controlling cell fate determination remain to be discovered in the anaerobic Clostridia.

The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health-care-associated diarrhea worldwide. Although C. difficile spore formation is essential for disease transmission, the regulatory pathways that control this developmental process have only been partially characterized. In the well-studied spore-former Bacillus subtilis, the highly conserved σ(E) , SpoIIID and σ(K) regulatory proteins control gene expression in the mother cell to ensure proper spore formation. To define the precise requirement for SpoIIID and σ(K) during C. difficile sporulation, we analyzed spoIIID and sigK mutants using heterologous expression systems and RNA-Seq transcriptional profiling. These analyses revealed that expression of sigK from a SpoIIID-independent promoter largely bypasses the need for SpoIIID to produce heat-resistant spores. We also observed that σ(K) is active upon translation, suggesting that SpoIIID primarily functions to activate sigK. SpoIIID nevertheless plays auxiliary roles during sporulation, as it enhances levels of the exosporium morphogenetic protein CdeC in a σ(K) -dependent manner. Analyses of purified spores further revealed that SpoIIID and σ(K) control the adherence of the CotB coat protein to C. difficile spores, indicating that these proteins regulate multiple stages of spore formation. Collectively, these results highlight that diverse mechanisms control spore formation in the Firmicutes.

The Gram-positive nosocomial pathogen Clostridium difficile induces sporulation during growth in the gastrointestinal tract. Sporulation is necessary for this obligate anaerobe to form metabolically dormant spores that can resist antibiotic treatment, survive exit from the mammalian host, and transmit C. difficile infections. In this chapter, we describe a method for inducing C. difficile sporulation in vitro. This method can be used to study sporulation and maximize spore purification yields for a number of C. difficile strain backgrounds. We also describe procedures for visualizing spore formation using phase-contrast microscopy and for quantifying the efficiency of sporulation using heat resistance as a measure of functional spore formation.

The Gram-positive nosocomial pathogen Clostridium difficile induces sporulation during growth in the gastrointestinal tract. Sporulation is necessary for this obligate anaerobe to form metabolically dormant spores that can resist antibiotic treatment, survive exit from the mammalian host, and transmit C. difficile infections. In this chapter, we describe a method for inducing C. difficile sporulation in vitro. This method can be used to study sporulation and maximize spore purification yields for a number of C. difficile strain backgrounds. We also describe procedures for visualizing spore formation using phase-contrast microscopy and for quantifying the efficiency of sporulation using heat resistance as a measure of functional spore formation.

The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health-care-associated diarrhea worldwide. Although C. difficile spore formation is essential for disease transmission, the regulatory pathways that control this developmental process have only been partially characterized. In the well-studied spore-former Bacillus subtilis, the highly conserved σ(E) , SpoIIID and σ(K) regulatory proteins control gene expression in the mother cell to ensure proper spore formation. To define the precise requirement for SpoIIID and σ(K) during C. difficile sporulation, we analyzed spoIIID and sigK mutants using heterologous expression systems and RNA-Seq transcriptional profiling. These analyses revealed that expression of sigK from a SpoIIID-independent promoter largely bypasses the need for SpoIIID to produce heat-resistant spores. We also observed that σ(K) is active upon translation, suggesting that SpoIIID primarily functions to activate sigK. SpoIIID nevertheless plays auxiliary roles during sporulation, as it enhances levels of the exosporium morphogenetic protein CdeC in a σ(K) -dependent manner. Analyses of purified spores further revealed that SpoIIID and σ(K) control the adherence of the CotB coat protein to C. difficile spores, indicating that these proteins regulate multiple stages of spore formation. Collectively, these results highlight that diverse mechanisms control spore formation in the Firmicutes.

Campylobacter jejuni is among the most common causes of enteric infection worldwide, and its complex relationship with the human host is just starting to be understood. C. jejuni causes inflammatory enteritis manifested by diarrhea or dysentery, fever, and abdominal cramping. Asymptomatic infection/colonization is also common, as described in children after repeated exposure to C. jejuni in resource-poor countries [1, 2]. Recently, incidence estimates of 1 symptomatic or asymptomatic C. jejuni infection every 2 years have also been reported in adults in developed countries [2]. C. jejuni infections have strong associations with postinfectious sequelae, strain variability, and increasing resistance to antibiotics. These include the demyelinating neurologic syndrome Guillain-Barre, chronic gastrointestinal symptoms, and postinfectious arthritis [3–6]. The kinetics and composition of the human immune response to C. jejuni are poorly understood and difficult to evaluate in field settings because of the inability to know onset of infection, strain differences, and previous exposures. Human challenge models, in contrast, provide a controlled method to understand and define immunologic responses to infection and/or correlates of protection [7–9]. We and others have described the human challenge model development of C. jejuni, most recently using strain CG8421 [9]. This strain, which lacks ganglioside mimicry in its lipo-oligosaccaharide, replaced strain 81-176, which expresses ganglioside 2 and ganglioside 3 and was epidemiologically linked to Guillain-Barre [7, 10]. Previous human challenge studies with 81-176 and A3249 demonstrated that Campylobacter-specific immunoglobulin A (IgA) and interferon γ (IFN-γ) are associated with resistance to clinical disease, suggesting these components might be important markers of protective immunity [7, 8]. To better define protective immunity to C. jejuni and to further develop the model, we challenged healthy, immunologically naive adults with C. jejuni CG8421 and then rechallenged subjects 3 months later with the same strain. As with previous challenge trials, this study was performed with the expectation that a primary infection would afford significant, if not complete, clinical protection after rechallenge [7, 8].

The Gram-positive, spore-forming pathogen Clostridium difficile is the leading definable cause of healthcare-associated diarrhea worldwide. C. difficile infections are difficult to treat because of their frequent recurrence, which can cause life-threatening complications such as pseudomembranous colitis. The spores of C. difficile are responsible for these high rates of recurrence, since they are the major transmissive form of the organism and resistant to antibiotics and many disinfectants. Despite the importance of spores to the pathogenesis of C. difficile, little is known about their composition or formation. Based on studies in Bacillus subtilis and other Clostridium spp., the sigma factors σF, σE, σG, and σK are predicted to control the transcription of genes required for sporulation, although their specific functions vary depending on the organism. In order to determine the roles of σF, σE, σG, and σK in regulating C. difficile sporulation, we generated loss-of-function mutations in genes encoding these sporulation sigma factors and performed RNA-Sequencing to identify specific sigma factor-dependent genes. This analysis identified 224 genes whose expression was collectively activated by sporulation sigma factors: 183 were σF-dependent, 169 were σE-dependent, 34 were σG-dependent, and 31 were σK-dependent. In contrast with B. subtilis, C. difficile σE was dispensable for σG activation, σG was dispensable for σK activation, and σF was required for post-translationally activating σG. Collectively, these results provide the first genome-wide transcriptional analysis of genes induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes., Author Summary C. difficile is the leading cause of healthcare-associated infectious diarrhea in the United States in large part because of its ability to form spores. Since spores are resistant to most disinfectants and antibiotics, C. difficile infections frequently recur and are easily spread. Despite the importance of spores to C. difficile transmission, little is known about how spores are made. We set out to address this question by generating C. difficile mutants lacking regulatory factors required for sporulation and identifying genes that are regulated by these factors during spore formation using whole-genome RNA-Sequencing. We determined that the regulatory pathway controlling sporulation in C. difficile differs from related Clostridium species and the non-pathogenic model spore-former Bacillus subtilis and identified 314 genes that are induced during C. difficile spore development. Collectively, our study provides a framework for identifying C. difficile gene products that are essential for spore formation. Further characterization of these gene products may lead to the identification of diagnostic biomarkers and the development of new therapeutics.

The four dengue virus serotypes (DENV-1–DENV-4) have a large impact on global health, causing 50–100 million cases of dengue fever annually. Herein, we describe the first kinetic T cell response to a low-dose DENV-1 vaccination study (10 PFU) in humans. Using flow cytometry, we found that proinflammatory cytokines, IFNγ, TNFα, and IL-2, were generated by DENV-1-specific CD4+ cells 21 days post-DENV-1 exposure, and their production continued through the latest time-point, day 42 (p, Author Summary 40% of the world's population is at risk for developing dengue fever, an acute febrile illness caused by the 4 serotypes of dengue viruses (DENV). Though most of the 50–100 million annual DENV infections resolve without medical intervention, approximately 500,000 cases are severe and require hospitalization. Supportive care is currently the only available treatment for dengue disease. As a result, DENV infections cause strain on healthcare systems and economic burden in endemic countries. Much of the research in the dengue field has focused on understanding the mechanism of severe dengue disease. To better understand human adaptive immune responses to asymptomatic or mild DENV infections, we used longitudinal specimens collected following low dose vaccination with a live DENV-1 candidate vaccine. We found that CD4+ T cells made the proinflammatory cytokines, IFNγ, TNFα and IL-2, 3 weeks following exposure to DENV-1. IFNγ and TNFα production continued for 6 weeks post-vaccination, our final time-point. T cell responses were predominantly multifunctional: T cells produced ≥2 cytokines simultaneously. Lastly, we observed little cross-reactivity in T cell responses. This work helps establish the kinetics and characteristics of a primary adaptive immune response to DENV and aids in the development of a tetravalent vaccine against DENV.

Although vaccines have been available for over a century, a correlate of protection for typhoid fever has yet to be identified. Antibodies are produced in response to typhoid infection and vaccination and are generally used as the gold standard for determining vaccine immunogenicity, even though their role in clearance of Salmonella enterica serovar Typhi infections is poorly defined. Here, we describe the first functional characterization of S. Typhi-specific antibodies following vaccination with a new vaccine, M01ZH09 (Ty2 Δ aroC Δ ssaV ). We determined that postvaccination sera increased the uptake of wild-type S. Typhi by human macrophages up to 2.3-fold relative to prevaccination (day 0) or placebo samples. These results were recapitulated using immunoglobulins purified from postvaccination serum, demonstrating that antibodies were largely responsible for increases in uptake. Imaging verified that macrophages internalized 2- to 9.5-fold more S. Typhi when the bacteria were opsonized with postvaccination sera than when the bacteria were opsonized with day 0 or placebo sera. Once inside macrophages, the survival of S. Typhi was reduced as much as 50% when opsonized with postvaccination sera relative to day 0 or placebo serum samples. Lastly, bactericidal assays indicated that antibodies generated postvaccination were recognized by complement factors and assisted in killing S. Typhi: mean postvaccination bactericidal antibody titers were higher at all time points than placebo and day 0 titers. These data clearly demonstrate that there are at least two mechanisms by which antibodies facilitate killing of S. Typhi. Future work could lead to improved immunogenicity tests associated with vaccine efficacy and the identification of correlates of protection against typhoid fever.