Jenn Wei Chen

Immunogenicity and protective efficacy of the Mycobacterium avium subsp. paratuberculosis attenuated mutants against challenge in a mouse model.

Johnes disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), results in serious economic losses worldwide especially in cattle, sheep and goats. To control the impact of JD on the animal industry, an effective vaccine with minimal adverse effects is urgently required. In order to develop an effective vaccine, we used allelic exchange to construct three mutant MAP strains, leuD, mpt64 and secA2. The mutants were attenuated in a murine model and induced cytokine responses in J774A.1 cell. The leuD mutant was the most obviously attenuated of the three constructed mutant strains. Our preliminary vaccine trial in mice demonstrated different levels of protection were induced by these mutants based on the acid-fast bacilli burden in livers and spleens at 8 and 12 weeks postchallenge. In addition, vaccination with leuD mutant induced a high level of IFN-γ production and significant protective efficacy in both the reduction of inflammation and clearance of acid-fast bacilli, as compared with the mock vaccinated group.

Immunostimulatory and antigen delivery properties of liposomes made up of total polar lipids from non-pathogenic bacteria leads to efficient induction of both innate and adaptive immune responses

Novel liposomes prepared from total polar lipids of non-pathogenic bacteria, viz. Leptospira biflexa serovar Potac (designated leptosomes) and Mycobacterium smegmatis (designated smegmosomes) were evaluated for their adjuvant effects with various antigen presenting cells (APCs), viz. murine macrophage cell line, J774A.1 and bone marrow derived dendritic cells (BMDCs). These liposomes induced strong membrane fusion as evident from resonance energy transfer (RET) assays and effectively transferred the fluorescent probe to the membrane of these APCs. Moreover, both vesicles caused significant activation of APCs as revealed by release of proinflammatory cytokines (IL-6, IL-12, TNF-α) and enhanced expression of co-stimulatory signals and maturation markers (CD80, CD86, MHCII), which was significantly higher for smegmosomes as compared to leptosomes. Additionally, activation of APCs by liposomes correlated with their ability to stimulate allospecific T cell proliferation and IFN-γ release. In contrast, conventional PC/chol liposomes failed to fuse and induced only a very low level of APC activation. Interestingly, the stimulatory activity of these lipid vesicles was restricted to APCs as they did not cause any significant activation or mitogenic effect on lymphocytes (B and T cells) in vitro. Overall, the activation of APCs by both leptosomes and smegmosomes correlated with activation of strong humoral and cell mediated immune responses in C57/BL6 mice to entrapped ovalbumin (OVA) and was significantly higher than those induced by conventional liposomes and alum, which failed to activate cytotoxic T lymphocytes (CTLs). Taken together these results demonstrate the adjuvant potential of these novel lipid vesicles that may simultaneously induce both innate and adaptive immune responses due to their immune stimulatory and antigen delivery properties.

Evaluation of a Mycobacterium avium subsp. paratuberculosis leuD Mutant as a Vaccine Candidate against Challenge in a Caprine Model

ABSTRACT Johnes disease (JD) is prevalent worldwide and has a significant impact on the global agricultural economy. In the present study, we evaluated the protective efficacy of a leuD (Δleud) mutant and gained insight into differential immune responses after challenge with virulent M. avium subsp. paratuberculosis in a caprine colonization model. The immune response and protective efficacy were compared with those of the killed vaccine Mycopar. In vitro stimulation of peripheral blood mononuclear cells with johnin purified protein derivative showed that Mycopar and ΔleuD generated similar levels of gamma interferon (IFN-γ) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with ΔleuD was the IFN-γ response maintained. Flow cytometric analysis showed that the increase in IFN-γ correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and γδT cells, but this response was significantly higher in ΔleuD-vaccinated animals at some time points after challenge. Both Mycopar and ΔleuD vaccines upregulated Th1/proinflammatory and Th17 cytokines and downregulated Th2/anti-inflammatory and regulatory cytokines at similar levels at almost all time points. However, significantly higher levels of IFN-γ (at weeks 26 and 30), interleukin-2 (IL-2; week 18), IL-1b (weeks 14 and 22), IL-17 (weeks 18 and 22), and IL-23 (week 18) and a significantly lower level of IL-10 (weeks 14 and 18) and transforming growth factor β (week 18) were detected in the ΔleuD-vaccinated group. Most importantly, ΔleuD elicited an immune response that significantly limited colonization of tissues compared to Mycopar upon challenge with wild-type M. avium subsp. paratuberculosis. In conclusion, the ΔleuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants.

Phenotypic and Transcriptomic Response of Auxotrophic Mycobacterium avium Subsp. paratuberculosis leuD Mutant under Environmental Stress

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of severe gastroenteritis in cattle. To gain a better understanding of MAP virulence, we investigated the role of leuD gene in MAP metabolism and stress response. For this, we have constructed an auxotrophic strain of MAP by deleting the leuD gene using allelic exchange. The wildtype and mutant strains were then compared for metabolic phenotypic changes using Biolog phenotype microarrays. The responses of both strains to physiologically relevant stress conditions were assessed using DNA microarrays. Transcriptomic data was then analyzed in the context of cellular metabolic pathways and gene networks. Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response. At the metabolic level, the mutant strain lost the ability to utilize most of the carbon, nitrogen, sulphur, phosphorus and nutrient supplements as energy source. At the transcriptome level, more than 100 genes were differentially expressed in each of the stress condition tested. Systems level network analysis revealed that the differentially expressed genes were distributed throughout the gene network, thus explaining the global impact of leuD deletion in metabolic phenotype. Further, we find that leuD deletion impacted metabolic pathways associated with fatty acids. We verified this by experimentally estimating the total fatty acid content of both mutant and wildtype. The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses. Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.

Comparative nutritional and chemical phenome of Clostridium difficile isolates determined using phenotype microarrays

OBJECTIVES Clostridium difficile infection (CDI) is the leading cause of infectious diarrhea in North America and Europe. The risk of CDI increases significantly in the case where antimicrobial treatment reduces the number of competing bacteria in the gut, thus leading to the increased availability of nutrients and loss of colonization resistance. The objective of this study was to determine comprehensive nutritional utilization and the chemical sensitivity profile of historic and newer C. difficile isolates and to examine the possible role of the phenotype diversity in C. difficile virulence. METHODS Phenotype microarrays (PMs) were used to elucidate the complete nutritional and chemical sensitivity profile of six C. difficile isolates. RESULTS Of the 760 nutrient sources tested, 285 compounds were utilized by at least one strain. Among the C. difficile isolates compared, R20291, a recent hypervirulent outbreak-associated strain, appears to have an expanded nutrient utilization profile when compared to all other strains. CONCLUSIONS The expanded nutritional utilization profile of some newer C. difficile strains could be one of the reasons for infections in patients who are not exposed to the hospital environment or not undergoing antibiotic treatment. This nutritional profile could be used to design tube feeding formulas that reduce the risk of CDI.

Differential stress transcriptome landscape of historic and recently emerged hypervirulent strains of Clostridium difficile strains determined using RNA-seq.

C. difficile is the most common cause of nosocomial diarrhea in North America and Europe. Genomes of individual strains of C. difficile are highly divergent. To determine how divergent strains respond to environmental changes, the transcriptomes of two historic and two recently isolated hypervirulent strains were analyzed following nutrient shift and osmotic shock. Illumina based RNA-seq was used to sequence these transcriptomes. Our results reveal that although C. difficile strains contain a large number of shared and strain specific genes, the majority of the differentially expressed genes were core genes. We also detected a number of transcriptionally active regions that were not part of the primary genome annotation. Some of these are likely to be small regulatory RNAs.

Immune response and protective efficacy of live attenuated Salmonella vaccine expressing antigens of Mycobacterium avium subsp. paratuberculosis against challenge in mice.

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants that leads to diarrhea and eventually death. Existing vaccines have proven useful in limiting disease progression but have not been effective in preventing infection. To address this problem we constructed an attenuated Salmonella (ΔyejE; ΔssaV) strain harboring a plasmid that expressed a fusion protein comprised of the Salmonella Type III secretion system (T3SS) effector SopE and MAP antigens (85A, 85B, SOD, 74F) and evaluated its potential as vaccine candidate against MAP infection in mice. Of various SopE-MAP fusion proteins analyzed, only SopE104-Ag85A C-terminal(202-347)-SOD N-terminal(1-72)-Ag85B C-terminal(173-330) and SopE104-74F(1-148+669-786)were successfully expressed and secreted into culture media as revealed by western blot analysis. Mice immunized with attenuated Salmonella (ΔyejE; ΔssaV) harboring the SopE104-Ag85A C-terminal(202-347)-SOD N-terminal(1-72)-Ag85B C-terminal(173-330) and SopE104-74F(1-148+669-786)plasmid generated a potent and long lasting Th1 response characterized by production of IFN-γ. The cytokine profile varied at various time points after immunization and challenge, which showed down regulation of Th2 cytokines (IL-4, IL-10) and up-regulation of proinflammatory cytokines (IL-12 and IL-17). Further, the immune response correlated with protection as revealed by reduced bacterial load and improved histopathology of spleen and liver, which showed fewer granulomas and lower numbers of acid-fast bacilli as compared to PBS controls. Interestingly, vaccination with antigens mixed with Ribi adjuvant (Agmix+Ribi) imparted better protection than the attenuated salmonella vectored vaccine. Thus, priming with a live recombinant Salmonella strain that secretes MAP antigens represents a promising approach that could lead to development of an efficacious and cost effective vaccine for Johnes disease.

Comparative genomic and phenomic analysis of Clostridium difficile and Clostridium sordellii , two related pathogens with differing host tissue preference

BackgroundClostridium difficile and C. sordellii are two anaerobic, spore forming, gram positive pathogens with a broad host range and the ability to cause lethal infections. Despite strong similarities between the two Clostridial strains, differences in their host tissue preference place C. difficile infections in the gastrointestinal tract and C. sordellii infections in soft tissues.ResultsIn this study, to improve our understanding of C. sordellii and C. difficile virulence and pathogenesis, we have performed a comparative genomic and phenomic analysis of the two. The global phenomes of C. difficile and C. sordellii were compared using Biolog Phenotype microarrays. When compared to C. difficile, C. sordellii was found to better utilize more complex sources of carbon and nitrogen, including peptides. Phenotype microarray comparison also revealed that C. sordellii was better able to grow in acidic pH conditions. Using next generation sequencing technology, we determined the draft genome of C. sordellii strain 8483 and performed comparative genome analysis with C. difficile and other Clostridial genomes. Comparative genome analysis revealed the presence of several enzymes, including the urease gene cluster, specific to the C. sordellii genome that confer the ability of expanded peptide utilization and survival in acidic pH.ConclusionsThe identified phenotypes of C. sordellii might be important in causing wound and vaginal infections respectively. Proteins involved in the metabolic differences between C. sordellii and C. difficile should be targets for further studies aimed at understanding C. difficile and C. sordellii infection site specificity and pathogenesis.

Mutation of the enterohemorrhagic escherichia coli core LPS biosynthesis enzyme RfaD confers hypersusceptibility to host intestinal innate immunity in vivo

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC, and LPS biosynthesis has long been considered as potential anti-bacterial target. Here, we demonstrated that the EHEC rfaD gene that functions in the biosynthesis of the LPS inner core is required for the intestinal colonization and pathogenesis of EHEC in vivo. Disruption of the EHEC rfaD confers attenuated toxicity in Caenorhabditis elegans and less bacterial colonization in the intestine of C. elegans and mouse. Moreover, rfaD is also involved in the control of susceptibility of EHEC to antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Taken together, we demonstrated that mutations of the EHEC rfaD confer hypersusceptibility to host intestinal innate immunity in vivo, and suggested that targeting the RfaD or the core LPS synthesis pathway may provide alternative therapeutic regimens for EHEC infection.

A multi-omic analysis reveals the role of fumarate in regulating the virulence of enterohemorrhagic Escherichia coli

The enteric pathogen enterohemorrhagic Escherichia coli (EHEC) is responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Several molecular mechanisms have been described for the pathogenicity of EHEC; however, the role of bacterial metabolism in the virulence of EHEC during infection in vivo remains unclear. Here we show that aerobic metabolism plays an important role in the regulation of EHEC virulence in Caenorhabditis elegans. Our functional genomic analyses showed that disruption of the genes encoding the succinate dehydrogenase complex (Sdh) of EHEC, including the sdhA gene, attenuated its toxicity toward C. elegans animals. Sdh converts succinate to fumarate and links the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) simultaneously. Succinate accumulation and fumarate depletion in the EHEC sdhA mutant cells were also demonstrated to be concomitant by metabolomic analyses. Moreover, fumarate replenishment to the sdhA mutant significantly increased its virulence toward C. elegans. These results suggest that the TCA cycle, ETC, and alteration in metabolome all account for the attenuated toxicity of the sdhA mutant, and Sdh catabolite fumarate in particular plays a critical role in the regulation of EHEC virulence. In addition, we identified the tryptophanase (TnaA) as a downstream virulence determinant of SdhA using a label-free proteomic method. We demonstrated that expression of tnaA is regulated by fumarate in EHEC. Taken together, our multi-omic analyses demonstrate that sdhA is required for the virulence of EHEC, and aerobic metabolism plays important roles in the pathogenicity of EHEC infection in C. elegans. Moreover, our study highlights the potential targeting of SdhA, if druggable, as alternative preventive or therapeutic strategies by which to combat EHEC infection.