Rui Huang

Salmonella pathogenicity island 1(SPI-1) at work.

As an indispensable virulence determinant, Salmonella pathogenicity island 1(SPI-1) has gained much attention in host–pathogen interactions. More and more studies on SPI-1 have demonstrated that it is far more complex than the one we used to expect. It not only affects sophisticated activities during infection, including invasion, replication, and host responses, but also extends to other fields like biofilm formation. In addition, mutants in SPI-1 effectors have been explored as vaccines, the effective ways to prevent salmonellosis. The activity of SPI-1 is influenced by many factors which have been widely reported. Investigations on these factors may provide new insights into prevention and treatment of salmonellosis. Therefore, SPI-1 is a tempting issue worthy of further exploration. In this review, we will probe into SPI-1 systematically.

Extracellular DNA inhibits Salmonella enterica Serovar Typhimurium and S. enterica Serovar Typhi biofilm development on abiotic surfaces.

AbstractnExtracellular DNA (eDNA) was identified and characterized in a 2-day-old biofilms developed by Salmonella enterica ser. Typhimurium SR-11 and S. enterica ser. Typhi ST6 using confocal laser scanning microscopy (CLSM) and enzymatic extraction methods. Results of microtitre plate assay and CLSM analysis showed both Salmonella strains formed significantly more biofilms in the presence of DNase I; Furthermore, a remarkable decrease of biofilm formation was observed when eDNA was added in the inoculation. However, for the pre-established biofilms on polystyrene and glass, no significant difference was observed between the DNase I treated biofilm and the corresponding non-treated controls. In conclusion, these results demonstrate that eDNA is a novel matrix component of Salmonella biofilms. This is the first evidence for the presence of eDNA and its inhibitive and destabilizing effect during biofilm development of S. enterica ser. Typhimurium and S. enterica ser. Typhi on abiotic surfaces.

Salmonella plasmid virulence gene spvB enhances bacterial virulence by inhibiting autophagy in a zebrafish infection model.

Salmonella enterica serovar typhimurium (S.xa0typhimurium) is a facultative intracellular pathogen that can cause gastroenteritis and systemic infection in a wide range of hosts. Salmonella plasmid virulence gene spvB is closely related to bacterial virulence in different cells and animal models, and the encoded protein acts as an intracellular toxin required for ADP-ribosyl transferase activity. However, until now there is no report about the pathogenecity of spvB gene on zebrafish. Due to the outstanding advantages of zebrafish in analyzing bacteria-host interactions, a S.xa0typhimurium infected zebrafish model was set up here to study the effect of spvB on autophagy and intestinal pathogenesis inxa0vivo. We found that spvB gene could decrease the LD50 of S.xa0typhimurium, and the strain carrying spvB promoted bacterial proliferation and aggravated the intestinal damage manifested by the narrowed intestines, fallen microvilli, blurred epithelium cell structure and infiltration of inflammatory cells. Results demonstrated the enhanced virulence induced by spvB in zebrafish. In spvB-mutant strain infected zebrafish, the levels of Lc3 turnover and Beclin1 expression increased, and the double-membraned autophagosome structures were observed, suggesting that spvB can inhibit autophagy activity. In summary, our results indicate that S.xa0typhimurium strain containing spvB displays more virulence, triggering an increase in bacterial survival and intestine injuries by suppressing autophagy for the first time. This model provides novel insights into the role of Salmonella plasmid virulence gene in bacterial pathogenesis, and can help to further elucidate the relationship between bacteria and host immune response.

Salmonella spv locus suppresses host innate immune responses to bacterial infection

Salmonella enterica serovar typhimurium (S. typhimurium) is globally distributed and causes massive morbidity and mortality in humans and animals. S. typhimurium carries Salmonella plasmid virulence (spv) locus, which is highly conserved and closely related to bacterial pathogenicity, while its exact role in host immune responses during infection remains to be elucidated. To counteract the invaders, the host has evolved numerous strategies, among which the innate immunity and autophagy act as the first defense. Recently, zebrafish has been universally accepted as a valuable and powerful vertebrate model in analyzing bacteria-host interactions. To investigate whether spv locus enhances the virulence of Salmonella by exerting an effect on the host early defense, zebrafish larvae were employed in this study. LD50 of S. typhimurium to zebrafish larvae and bacterial dissemination were analyzed. Sudan black B and neutral red staining were performed to detect the responses of neutrophils and macrophages to Salmonella infection. Autophagy agonist Torin1 and inhibitor Chloroquine were used to interfere in autophagic flux, and the protein level of Lc3 and p62 were measured by western blotting. Results indicated that spv locus could decrease the LD50 of S. typhimurium to zebrafish larvae, accelerate the reproduction and dissemination of bacteria by inhibiting the function of neutrophils and macrophages. Moreover, spv locus restrained the formation of autophagosomes in the earlier stage of autophagy. These findings suggested the virulence of spv locus involving in suppressing host innate immune responses for the first time, which shed new light on the role of spv operon in Salmonella pathogenicity.

Salmonella enterica serovar Typhi plasmid impairs dendritic cell responses to infection.

Salmonella enterica serovar Typhi (S. typhi) evades from innate immunity by expression of a variety of pathogenic factors. The “pRST98” plasmid of S. typhi is involved in multidrug-resistant and virulence of S. typhi. However, its exact effect on host cell function remains elusive. Dendritic cells (DCs) play an important role in shaping immune response against Salmonella. For the purpose of investigation whether pRST98 might target DCs involved in adaptive immune response, murine DCs were infected with S. typhi wild type and mutant strains. S. typhi stimulation resulted in up-regulation of costimulatory molecules on DCs. S. typhi wild type resulted in decreased up-regulation of CD40, CD80, and CD86 expression. Experiments with S. typhi pRST98 mutant (S. typhi-Δ-pRST98) and S. typhi-Δ-pRST98 with a complemented plasmid encoding pRST98 (S. typhi-c-pRST98) revealed that pRST98 accounts for inhibition of surface molecule expression and functional maturity. S. typhi-Δ-pRST98 gave maximal levels of IL-12 and IFN-γ release compared with wild type S. typhi or the complemented strains. In contrast to IL-12 and IFN-γ, IL-10 secretion by S.typhi-Δ-pRST98-infected DCs was significantly lower than induction by S.typhi wild type. This indicates that immunity in response to pRST98 is skewed away from a protective Th1 response. Moreover, infection with S. typhi-Δ-pRST98 induced autophagy in DCs. We herein demonstrate S. typhi pRST98 plays essential roles in modulating DCs maturation, activation, inflammatory responses, and autophagy. Together, these data prove that pRST98 targets functions of DCs that are required for T-cell activation. This might contribute to evasion of adaptive immune responses by S. typhi.

A novel contribution of spvB to pathogenesis of Salmonella Typhimurium by inhibiting autophagy in host cells

Salmonella plasmid virulence genes (spv) are highly conserved in strains of clinically important Salmonella serovars. It is essential for Salmonella plasmid-correlated virulence, although the exact mechanism remains to be elucidated. Autophagy has been reported to play an important role in host immune responses limiting Salmonella infection. Our previous studies demonstrated that Salmonella conjugative plasmid harboring spv genes could enhance bacterial cytotoxicity by inhibiting autophagy. In the present study, we investigated whether spvB, which is one of the most important constituents of spv ORF could intervene in autophagy pathway. Murine macrophage-like cells J774A.1, human epithelial HeLa cells, and BALB/c mice infected with Salmonella Typhimurium wild type, mutant and complementary strains (carrying or free spvB or complemented only with ADP-ribosyltransferase activity of SpvB) were used in vitro and in vivo assay, respectively. To further explore the molecular mechanisms, both SpvB ectopic eukaryotic expression system and cells deficient in essential autophagy components by siRNA were generated. Results indicated that spvB could suppress autophagosome formation through its function in depolymerizing actin, and aggravate inflammatory injury of the host in response to S. Typhimurium infection. Our studies demonstrated virulence of spvB involving in inhibition of autophagic flux for the first time, which could provide novel insights into Salmonella pathogenesis, and have potential application to develop new antibacterial strategies for Salmonellosis.

A Salmonella enterica conjugative plasmid impairs autophagic flux in infected macrophages

pR(ST98) was originally isolated from Salmonella enterica serovar typhi and could be transferred among enteric bacilli by conjugation. Our previous studies indicated that it could intervene in autophagy of host cells, while the mechanism remained undefined. Here, we explored how pR(ST98) influenced the autophagic flux of murine macrophage-like cell line (J774A.1). S. enterica serovar typhimurium wild type strain (χ3306), harboring a 100 kb virulence plasmid, was used as a positive control. pR(ST98) was transferred into χ3306 virulence plasmid cured strain (χ3337) to create the transconjugant strain (χ3337/pR(ST98)). The bacterial strains incubated with J774A.1 revealed that survival rate of intracellular bacteria carrying pR(ST98) was higher than that of plasmid free strain; presence of pR(ST98) decreased the number of autophagy vacuoles, LC3 positive and p62 positive bacteria, and also the level of LC3-II and degradation of p62 in macrophages. After intervention with autophagy inhibitor chloroquine, the amount of LC3-II and autophagy vacuoles were still lower in macrophages infected with strains carrying pR(ST98). Our study suggested that pR(ST98) could block or delay the formation of autophagosome in the earlier autophagy process, but couldnt affect the function of autolysosome. This finding provided novel insights into the role of enteric conjugation plasmid in bacterial pathogenesis.

Autophagy and Ubiquitination in Salmonella Infection and the Related Inflammatory Responses

Salmonellae are facultative intracellular pathogens that cause globally distributed diseases with massive morbidity and mortality in humans and animals. In the past decades, numerous studies were focused on host defenses against Salmonella infection. Autophagy has been demonstrated to be an important defense mechanism to clear intracellular pathogenic organisms, as well as a regulator of immune responses. Ubiquitin modification also has multiple effects on the host immune system against bacterial infection. It has been indicated that ubiquitination plays critical roles in recognition and clearance of some invading bacteria by autophagy. Additionally, the ubiquitination of autophagy proteins in autophagy flux and inflammation-related substance determines the outcomes of infection. However, many intracellular pathogens manipulate the ubiquitination system to counteract the host immunity. Salmonellae interfere with host responses via the delivery of ~30 effector proteins into cytosol to promote their survival and proliferation. Among them, some could link the ubiquitin-proteasome system with autophagy during infection and affect the host inflammatory responses. In this review, novel findings on the issue of ubiquitination and autophagy connection as the mechanisms of host defenses against Salmonella infection and the subverted processes are introduced.

spv locus aggravates Salmonella infection of zebrafish adult by inducing Th1/Th2 shift to Th2 polarization

Abstract Salmonella enterica serovar typhimurium (S. typhimurium) are facultative intracellular enteric pathogens causing disease with a broad range of hosts. It was known that Th1‐type cytokines such as IFN‐&ggr;, IL‐12, and TNF‐&agr; etc. could induce protective immunity against intracellular pathogens, while Th2‐type cytokines such as IL‐4, IL‐10, and IL‐13 etc. are proved to help pathogens survive inside hosts and cause severe infection. One of the critical virulence factor attributes to the pathogenesis of S. typhimurium is Salmonella plasmid virulence genes (spv). Until now, the interaction between spv locus and the predictable generation of Th1 or Th2 immune responses to Salmonella has not been identified. In this study, zebrafish adults were employed to explore the effect of spv locus on Salmonella pathogenesis as well as host adaptive immune responses especially shift of Th1/Th2 balance. The pathological changes of intestines and livers in zebrafish were observed by hematoxylin‐eosin (HE) staining and electron microscopy. Levels of the transcription factors of Th1 (Tbx21) and Th2 (GATA3) were measured by real‐time quantitative PCR (RT‐qPCR). Expression of cytokines were determined by using RT‐qPCR and ELISA, respectively. Results showed that spv operon aggravates damage of zebrafish. Furthermore, it demonstrated that spv locus could inhibit the transcription of tbx21 gene and suppress the expression of cytokines IFN‐&ggr;, IL‐12 and TNF‐&agr;. On the contrary, the transcription of gata3 gene could be promoted and the expression of cytokines IL‐4, IL‐10 and IL‐13 were enhanced by spv locus. Taken together, our data revealed that spv locus could aggravate Salmonella infection of zebrafish adult by inducing an imbalance of Th1/Th2 immune response and resulting in a detrimental Th2 bias of host. HighlightsSalmonella‐zebrafish adult infection model was successfully established.spv locus aggravates Salmonella infection in zebrafish adult manifesting severe damages of intestine and liver.spv operon induces an imbalance of Th1/Th2 immune response and results in a detrimental Th2 bias of host.