An indirect enzyme-linked immunosorbent assay for rapid and quantitative assessment of Type III virulence phenotypes of Pseudomonas aeruginosa isolates

Background

The presence of a Type III secretion system in clinical isolates of Pseudomonas aeruginosa is associated with severe disease and poor outcomes in infections caused by this pathogen. We describe an indirect enzyme-linked immunosorbent assay that rapidly and quantitatively detects two exotoxins, ExoU and ExoT, and two structural components, PopD and PcrV, of the P. aeruginosa Type III secretion system after in-vitro growth in a calcium-free minimal medium.

Methods

We used this assay to characterize the Type III secretion phenotype of 74 clinical isolates of P. aeruginosa. Findings were compared with results of standard immunoblotting and correlated with Type III secretion-dependent virulence of isolates toward cultured epithelial cells.

Results

Results of the ELISA assay were concordant with immunoblot detection of the secreted antigens for 73 of 74 isolates. The Type III secretion phenotype assessed by this immunoassay predicted bacterial virulence toward epithelial cells in vitro for all but five of the clinical isolates.

Conclusion

The availability of an ELISA assay for rapid detection of Type III secreted virulence factors will facilitate large clinical studies to examine whether the Type III secretion phenotype of a P. aeruginosa isolate predicts the course of clinical disease in a patient and should be taken into account in determining optimal treatment strategies for infected patients.     

Global Impact of Salmonella Pathogenicity Island 2-secreted Effectors on the Host Phosphoproteome

During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.Type III secretion systems (T3SSs)¹ are specialized virulence factors in Gram-negative pathogens that play an important role in delivering effector proteins to host cells. Salmonella enterica employs two distinct T3SSs encoded in Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2), with numerous effectors encoded around the genome, including a small number in SPI1 and SPI2 (1). SPI1 T3SS effectors are required for the bacterial internalization by intestinal epithelial cells at early stages of infection after oral ingestion. Although Salmonella is subsequently taken up by intestinal macrophages via phagocytosis, SPI2 T3SS effectors function to promote intracellular replication. Part of the role of SPI2 effectors is to control the maturation of the membrane-enclosed, Salmonella-containing vacuole (SCV) where Salmonella survives and replicates, eventually leading to a systemic infection known as typhoid fever (2, 3).Approximately 30 effectors are known to be translocated by the SPI2 T3SS but the actions and targets of most of these effectors are largely unknown (1, 3, 4). A recent systematic study using a single mutant collection of SPI2 genes showed particular virulence factors (e.g. SpvB, SifA, and SteC) play a dominant role in replication within macrophages (5). It is known that SpvB induces cytotoxicity through its ADP-ribosyltransferase activity (6), and SifA is required for maturation of the SCV and the formation of Salmonella-induced filaments (7). SteC has been identified as the sole serine/threonine protein kinase encoded in the Salmonella genome (8), but the target substrates of this kinase within the host are not fully understood, although it has been demonstrated that SteC partially targets the MAP kinase MEK (9). Interestingly, SteC is capable of promoting assembly of an F-actin meshwork around the SCV; this is dependent on its kinase activity but does not require activation of signaling pathways through Rho-associated protein kinase (8), Cdc42, Rac, N-WASP, Scar/WAVE, and Arp2/3 (10). These host signaling proteins are the main targets of T3SS-secreted effectors from many pathogens, including the SPI1 system in Salmonella (11) and Shigella (12). Therefore, SteC is thought to manipulate actin in a unique way through phosphorylation of host protein target(s).Recent advances in high throughput measurements allow us to characterize host gene expression profiles (13) and host phosphoproteme dynamics (14) dependent on the presence of SPI1 effectors in an unbiased, comprehensive manner. However, although it is clear that SPI2 T3SS is a major virulence factor contributing to systemic infection, our knowledge of its effects on host responses is limited. In this study, we used a mass spectrometry (MS)-based quantitative proteomics approach and measured global host phosphorylation changes as well as proteome abundance altered by SPI2 effectors. Furthermore, we explore a molecular target of SPI2 effector kinase SteC by integrating the phosphoproteomics data and other quantitative proteomics screens.     

Crystal Structure of the Salmonella enterica Serovar Typhimurium Virulence Factor SrfJ,a Glycoside Hydrolase Family Enzyme

To cause infection, Salmonella enterica serovar Typhimurium uses type III secretion systems, which are encoded on two Salmonella pathogenicity islands, SPI-1 and SPI-2, the latter of which is thought to play a crucial role in bacterial proliferation in Salmonella-containing vacuoles (SCVs) after invading cells. S. Typhimurium SrfJ, located outside SPI-2, is also known to be involved in Salmonella pathogenicity and has high amino acid sequence homology with human lysosomal glucosylceramidase (GlcCerase). We present the first crystal structure of SrfJ at a resolution of 1.8 Å. The overall fold of SrfJ shares high structure similarities with that of human GlcCerase, comprising two distinctive domains: a (β/α)₈-barrel catalytic domain and a β-sandwich domain. As in human GlcCerase, the pocket-shaped active site of SrfJ is located on the C-terminal side of the barrel, and two conserved glutamic acid residues are used for the enzyme catalysis. Moreover, a glycerol-bound form of SrfJ reveals that the glucose ring moiety of the substrate might similarly bind to the enzyme as to human GlcCerase, suggesting that SrfJ might function as a glycoside hydrolase. Although some structural differences are observed between SrfJ and human GlcCerase in the substrate entrance of the active site, we speculate that, based on the high structural similarities to human GlcCerase in the overall fold and the active-site environment, SrfJ might have a GlcCerase activity and use the activity to enhance Salmonella virulence by modifying SCV membrane lipids.Gram-negative bacterial pathogens deliver effector proteins into host cells through type III secretion systems (TTSS). The TTSS apparatus is a molecular syringe which spans the inner and outer membranes of pathogens and secretes translocon and effector proteins. Translocon proteins locate at the tip of the needle structure and are involved in the translocation of effector proteins by forming pores in the host cell membrane (3). The translocated effector proteins function to manipulate diverse host cellular processes such as cytoskeleton assembly, vesicle transport, and signal transduction, thereby promoting bacterial virulence (9).Salmonella enteric serovar Typhimurium (S. Typhimurium) causes a systemic infection in mice and is an intensively studied model of typhoid fever. This gram-negative bacterium can invade host cells and then survive by replicating within a membrane-bound compartment known as the Salmonella-containing vacuole (SCV) (16). Both invasion and intracellular survival are mediated by numerous virulence genes, which are clustered within the pathogenicity islands, SPI-1 and SPI-2 (18). The regulation of virulence proteins encoded by each pathogenicity island depends on the different stages of infection. While most of the genes within SPI-1 are required for the invading host cells and the early stages of SCV development (8), the genes in SPI-2 play a crucial role in bacterial proliferation in SCVs after cell invasion (23). The SsrA-SsrB two-component regulatory system is known to regulate the expression of genes within SPI-2 for bacterial virulence (4). Recent works have shown that several genes located outside of SPI-2 are under the control of the SsrA-SsrB regulator as well, and these have been proposed as putative virulence factors (10, 25).S. Typhimurium SrfJ was initially identified as a gene that is strongly activated by SsrB outside SPI-2 (25). Furthermore, a mutation on srfJ leads to mild attenuation of virulence in mice (22). Interestingly, SrfJ shares high amino acid sequence similarity with human lysosomal glucosylceramidase (GlcCerase) (25), which is a peripheral membrane protein catalyzing the hydrolysis of glucosylceramide (GlcCer) to β-glucose and ceramide in the presence of the modulator protein saposin C and lipid (11). Inherited defects in GlcCerase result in lysosomal GlcCer accumulation and, as a consequence, Gaucher disease, the most common lysosomal storage disease (19). Both human GlcCerase and SrfJ have been grouped into glycoside hydrolase (GH) family 30 containing GlcCerase (EC 3.2.1.45), β-1,6-glucanse (EC 3.2.1.75), and β-xylosidase (EC 3.2.1.37) of the GH-A clan in the CAZy database (http://afmb.cnrs-mrs.fr/CAZY). Among the members of GH family 30, structural information is available only on the human enzyme. The biochemical function of SrfJ and its role in Salmonella virulence remain to be elucidated. In order to better understand the function of SrfJ, we have determined the crystal structure of SrfJ from S. Typhimurium at a resolution of 1.8 Å.     

A novel anti-virulence gene revealed by proteomic analysis in Shigella flexneri 2a

Background

Shigella flexneri is a gram-negative, facultative pathogen that causes the majority of communicable bacterial dysenteries in developing countries. The virulence factors of S. flexneri have been shown to be produced at 37 degrees C but not at 30 degrees C. To discover potential, novel virulence-related proteins of S. flexneri, we performed differential in-gel electrophoresis (DIGE) analysis to measure changes in the expression profile that are induced by a temperature increase.

Results

The ArgT protein was dramatically down-regulated at 37 degrees C. In contrast, the ArgT from the non-pathogenic E. coli did not show this differential expression as in S. flexneri, which suggested that argT might be a potential anti-virulence gene. Competitive invasion assays in HeLa cells and in BALB/c mice with argT mutants were performed, and the results indicated that the over-expression of ArgT_Y225D would attenuate the virulence of S. flexneri. A comparative proteomic analysis was subsequently performed to investigate the effects of ArgT in S. flexneri at the molecular level. We show that HtrA is differentially expressed among different derivative strains.

Conclusion

Gene argT is a novel anti-virulence gene that may interfere with the virulence of S. flexneri via the transport of specific amino acids or by affecting the expression of the virulence factor, HtrA.     

E. coli Nissle 1917 Affects Salmonella adhesion to porcine intestinal epithelial cells

Background

The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known.

Methodology/Principal Findings

In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN.

Conclusions

We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion.     

Functional analysis of the group A streptococcal luxS/AI-2 system in metabolism, adaptation to stress and interaction with host cells

Background

The luxS/AI-2 signaling pathway has been reported to interfere with important physiological and pathogenic functions in a variety of bacteria. In the present study, we investigated the functional role of the streptococcal luxS/AI-2 system in metabolism and diverse aspects of pathogenicity including the adaptation of the organism to stress conditions using two serotypes of Streptococcus pyogenes, M1 and M19.

Results

Exposing wild-type and isogenic luxS-deficient strains to sulfur-limited media suggested a limited role for luxS in streptococcal activated methyl cycle metabolism. Interestingly, loss of luxS led to an increased acid tolerance in both serotypes. Accordingly, luxS expression and AI-2 production were reduced at lower pH, thus linking the luxS/AI-2 system to stress adaptation in S. pyogenes. luxS expression and AI-2 production also decreased when cells were grown in RPMI medium supplemented with 10% serum, considered to be a host environment-mimicking medium. Furthermore, interaction analysis with epithelial cells and macrophages showed a clear advantage of the luxS-deficient mutants to be internalized and survive intracellularly in the host cells compared to the wild-type parents. In addition, our data revealed that luxS influences the expression of two virulence-associated factors, the fasX regulatory RNA and the virulence gene sibA (psp).

Conclusion

Here, we suggest that the group A streptococcal luxS/AI-2 system is not only involved in the regulation of virulence factor expression but in addition low level of luxS expression seems to provide an advantage for bacterial survival in conditions that can be encountered during infections.     

HLA-B27 modulates intracellular growth of Salmonella pathogenicity island 2 mutants and production of cytokines in infected monocytic U937 cells

Background

Salmonella enterica serovar Enteritidis PT4 KS8822/88 replicates rapidly in HLA-B27-transfected human monocytic U937 cells. In this process, Salmonella pathogenicity island 2 (SPI-2) genes play a crucial role. Our previous study indicated that 118 Salmonella genes, including 8 SPI-2 genes were affected by HLA-B27 antigen during Salmonella infection of U937 cells.

Methods/Principal Findings

To further investigate Salmonella replication in HLA-B27-positive U937 monocytic cells, two SPI-2 genes, ssaS and sscA up-regulated most during Salmonella infection of HLA-B27-transfected U937 cells, were mutated by using one-step gene disruption method. Intracellular survival and replication of the mutants in the U937 cells was compared to that of the wild type strain. Surprisingly, the two mutated strains replicated significantly more than the wild type bacteria in HLA-B27-transfected cells. Secretion of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) was significantly induced during the infection of HLA-B27-transfected U937 cells with the mutants. The results indicated that the certain SPI-2 genes in wild type bacteria suppress Salmonella intracellular growth and production of cytokines in infected HLA-B27-transfected cells. HLA-B27-associated modulation of Salmonella SPI-2 genes and cytokine production may have importance in the persistent infection of the bacteria and the pathogenesis of reactive arthritis.

Conclusions

The study provides evidence that certain virulence factors of pathogens can reduce the intracellular growth in the host cells. We suggest that the limiting intracellular growth might be a strategy for persistence of bacteria in host cells, keeping a balance between pathogenic growth and pathogenesis.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

Oral infection with the <Emphasis Type="Italic">Salmonella enterica</Emphasis>serovar Gallinarum 9R attenuated live vaccine as a model to characterise immunity to fowl typhoid in the chicken

Background

Salmonella enterica serovar Gallinarum (S. Gallinarum) is the causative agent of fowl typhoid, a severe systemic disease of chickens that results in high mortality amongst infected flocks. Due to its virulence, the immune response to S. Gallinarum is poorly characterised. In this study we have utilised infection by the live attenuated S. Gallinarum 9R vaccine strain in inbred chickens to characterise humoral, cellular and cytokine responses to systemic salmonellosis.

Results

Infection with 9R results in a mild systemic infection. Bacterial clearance at three weeks post infection coincides with increases in circulating anti-Salmonella antibodies, increased T cell proliferation to Salmonella challenge and increased expression of interferon gamma. These responses peak at four weeks post infection, then decline. Only modest increases of expression of the pro-inflammatory cytokine interleukin-1β were detected early in the infection.

Conclusion

Infection of chickens with the 9R vaccine strain induces a mild form of systemic salmonellosis. This induces both cellular and humoral immune responses, which peak soon after bacterial clearance. Unlike enteric-associated Salmonella infections the immune response is not prolonged, reflecting the absence of persistence of Salmonella in the gastrointestinal tract. The findings here indicate that the use of the S. Gallinarum 9R vaccine strain is an effective model to study immunity to systemic salmonellosis in the chicken and may be employed in further studies to determine which components of the immune response are needed for protection.

     

Proteomes of Host Cell Membranes Modified by Intracellular Activities of Salmonella enterica

Intracellular pathogens need to establish a growth-stimulating host niche for survival and replication. A unique feature of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium is the creation of extensive membrane networks within its host. An understanding of the origin and function of these membranes is crucial for the development of new treatment strategies. However, the characterization of this compartment is very challenging, and only fragmentary knowledge of its composition and biogenesis exists. Here, we describe a new proteome-based approach to enrich and characterize Salmonella-modified membranes. Using a Salmonella mutant strain that does not form this unique membrane network as a reference, we identified a high-confidence set of host proteins associated with Salmonella-modified membranes. This comprehensive analysis allowed us to reconstruct the interactions of Salmonella with host membranes. For example, we noted that Salmonella redirects endoplasmic reticulum (ER) membrane trafficking to its intracellular niche, a finding that has not been described for Salmonella previously. Our system-wide approach therefore has the potential to rapidly close gaps in our knowledge of the infection process of intracellular pathogens and demonstrates a hitherto unrecognized complexity in the formation of Salmonella host niches.Bacterial pathogens have evolved sophisticated mechanisms enabling them to invade, reside in, and proliferate in a large range of eukaryotic hosts. This often involves hijacking the host phagosomal system, interfering with the host cell signaling and trafficking machinery, and establishing a replication niche to avoid clearance (1). Whereas some pathogens escape phagosomes and replicate in the host cytoplasm, most of the described pathogens replicate in membrane-bound, vacuole-like compartments (2). Such intracellular niches of various pathogens are diverse, and biogenesis often depends on the delivery of bacterial effector proteins into the host cell cytoplasm.Salmonella enterica, the causative agent of localized gastroenteritis and the life-threatening systemic infection known as typhoid fever, forms so-called Salmonella-containing vacuoles (SCVs)¹ inside host cells (3). SCVs mature through continuous interactions with endocytic and recycling pathways, accompanied by a spatial shift from the side of internalization to the juxtanuclear position close to the microtubule-organizing center (4, 5). Whereas the initial maturation steps are similar to the canonical phagosome biogenesis, the formation of an extensive tubular membrane network extending from the mature SCV is unique to Salmonella-infected host cells. This network contains various tubular structures such as Salmonella-induced filaments (SIFs), sorting nexin tubules, Salmonella-induced secretory carrier membrane protein 3 tubules, and lysosome-associated membrane protein 1-negative tubules (5–7), distinguishable by individual organelle marker proteins. For instance, tubules decorated with lysosome-associated membrane protein 1 (LAMP1) are known as SIFs (8, 9). In this paper we refer to all host membranes modified by intracellular Salmonella as Salmonella-modified membranes (SMMs).In general, the appearance of SMMs coincides with the onset of bacterial replication, and both phenomena are dependent on the translocation of effector proteins of the Salmonella Pathogenicity Island 2 (SPI2)-encoded type III secretion system (T3SS) (10, 11). These effector proteins manipulate a large number of host cell processes and force the host cell to create a suitable microenvironment for Salmonella (7, 12, 13). Although many Salmonella effector proteins have been described (14), much less is known about the host proteins that are manipulated to foster bacterial growth.A systematic proteome-wide analysis would be an important step toward understanding the mechanisms used by Salmonella to reorganize the host cell endosomal system during intracellular proliferation. However, one major challenge is the need to distinguish host proteins directed toward the Salmonella-induced compartments from those that are present independent of an infection.In this report we describe a novel method for the enrichment of SMMs and utilize a comparative strategy to identify proliferation-relevant host proteins. This systematic characterization of the SMM proteome provides new insights into the cellular origin and biogenesis of SMMs and identifies host cell proteins modified by the activity of intracellular Salmonella.     

First insight into Mycobacterium tuberculosis genetic diversity in Paraguay

Background

Francisella tularensis is a gram negative, facultative intracellular bacterium that is the etiological agent of tularemia. F. novicida is closely related to F. tularensis but has low virulence for humans while being highly virulent in mice. IglA is a 21 kDa protein encoded by a gene that is part of an iglABCD operon located on the Francisella pathogenicity island (FPI).

Results

Bioinformatics analysis of the FPI suggests that IglA and IglB are components of a newly described type VI secretion system. In this study, we showed that IglA regulation is controlled by the global regulators MglA and MglB. During intracellular growth IglA production reaches a maximum at about 10 hours post infection. Biochemical fractionation showed that IglA is a soluble cytoplasmic protein and immunoprecipitation experiments demonstrate that it interacts with the downstream-encoded IglB. When the iglB gene was disrupted IglA could not be detected in cell extracts of F. novicida, although IglC could be detected. We further demonstrated that IglA is needed for intracellular growth of F. novicida. A non-polar iglA deletion mutant was defective for growth in mouse macrophage-like cells, and in cis complementation largely restored the wild type macrophage growth phenotype.

Conclusion

The results of this study demonstrate that IglA and IglB are interacting cytoplasmic proteins that are required for intramacrophage growth. The significance of the interaction may be to secrete effector molecules that affect host cell processes.     

Mycophenolate mofetil inhibits the development of Coxsackie B3-virus-induced myocarditis in mice

Background

Streptococcus pneumoniae possesses large zinc metalloproteinases on its surface. To analyse the importance in virulence of three of these metalloproteinases, intranasal challenge of MF1 outbred mice was carried out using a range of infecting doses of wild type and knock-out pneumococcal mutant strains, in order to compare mice survival.

Results

Observation of survival percentages over time and detection of LD₅₀s of knock out mutants in the proteinase genes in comparison to the type 4 TIGR4 wild type strain revealed two major aspects: i) Iga and ZmpB, present in all strains of S. pneumoniae, strongly contribute to virulence in mice; (ii) ZmpC, only present in about 25% of pneumococcal strains, has a lower influence on virulence in mice.

Conclusions

These data suggest Iga, ZmpB and ZmpC as candidate surface proteins responsible for pneumococcal infection and potentially involved in distinct stages of pneumococcal disease.     

An incomplete TCA cycle increases survival of Salmonella Typhimurium during infection of resting and activated murine macrophages

Background

In comparison to the comprehensive analyses performed on virulence gene expression, regulation and action, the intracellular metabolism of Salmonella during infection is a relatively under-studied area. We investigated the role of the tricarboxylic acid (TCA) cycle in the intracellular replication of Salmonella Typhimurium in resting and activated macrophages, epithelial cells, and during infection of mice.

Methodology/Principal Findings

We constructed deletion mutations of 5 TCA cycle genes in S. Typhimurium including gltA, mdh, sdhCDAB, sucAB, and sucCD. We found that the mutants exhibited increased net intracellular replication in resting and activated murine macrophages compared to the wild-type. In contrast, an epithelial cell infection model showed that the S. Typhimurium ΔsucCD and ΔgltA strains had reduced net intracellular replication compared to the wild-type. The glyoxylate shunt was not responsible for the net increased replication of the TCA cycle mutants within resting macrophages. We also confirmed that, in a murine infection model, the S. Typhimurium ΔsucAB and ΔsucCD strains are attenuated for virulence.

Conclusions/Significance

Our results suggest that disruption of the TCA cycle increases the ability of S. Typhimurium to survive within resting and activated murine macrophages. In contrast, epithelial cells are non-phagocytic cells and unlike macrophages cannot mount an oxidative and nitrosative defence response against pathogens; our results show that in HeLa cells the S. Typhimurium TCA cycle mutant strains show reduced or no change in intracellular levels compared to the wild-type [1]. The attenuation of the S. Typhimurium ΔsucAB and ΔsucCD mutants in mice, compared to their increased net intracellular replication in resting and activated macrophages suggest that Salmonella may encounter environments within the host where a complete TCA cycle is advantageous.     

Salmonella Type III Secretion Effector SlrP Is an E3 Ubiquitin Ligase for Mammalian Thioredoxin

Salmonella enterica encodes two virulence-related type III secretion systems in Salmonella pathogenicity islands 1 and 2, respectively. These systems mediate the translocation of protein effectors into the eukaryotic host cell, where they alter cell signaling and manipulate host cell functions. However, the precise role of most effectors remains unknown. Using a genetic screen, we identified the small, reduction/oxidation-regulatory protein thioredoxin as a mammalian binding partner of the Salmonella effector SlrP. The interaction was confirmed by affinity chromatography and coimmunoprecipitation. In vitro, SlrP was able to mediate ubiquitination of ubiquitin and thioredoxin. A Cys residue conserved in other effectors of the same family that also possess E3 ubiquitin ligase activity was essential for this catalytic function. Stable expression of SlrP in HeLa cells resulted in a significant decrease of thioredoxin activity and in an increase of cell death. The physiological significance of these results was strengthened by the finding that Salmonella was able to trigger cell death and inhibit thioredoxin activity in HeLa cells several hours post-infection. This study assigns a functional role to the Salmonella effector SlrP as a binding partner and an E3 ubiquitin ligase for mammalian thioredoxin.Protein secretion is a basic function in all groups of bacteria. Many secretion systems have been found in Gram-negative bacteria, from the relatively simple type I secretion systems to the complex type III or type IV machines or the recently described type VI systems (reviewed in Refs. 1 and 2). Many pathogenic or symbiotic Gram-negative bacteria rely on type III secretion systems (T3SS)² for their interaction with host organisms. The T3SS is a protein export pathway that spans the inner membrane, periplasmic space, outer membrane, and host cell membrane. These complex structures are related to flagella and consist of at least 20 different subunits that enable the bacteria to translocate substrates into the cytosol of the eukaryotic host cell (reviewed in Ref. 3). These systems have also been referred to as injectisomes or molecular needles (4).Proteins secreted and translocated into eukaryotic cells through T3SS are called “effectors.” These effectors have the ability to suppress host defense signaling. Effectors of plant pathogens may target salicylic acid- and abscisic acid-dependent defenses, host vesicle trafficking, or interfere with host RNA metabolism. Effectors from animal pathogens modify the cytoskeleton to facilitate bacterial entry, modulate Rho GTPases and NF-κB, inhibit the host inflammatory response, elicit death of immune cells, and disrupt both adaptative and innate immune responses (reviewed in Ref. 5).Salmonella enterica produces two distinct T3SS essential for virulence that are encoded by genes located in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), respectively. The SPI-1 T3SS secretes at least 16 proteins: AvrA, GogB, SipA, SipB, SipC, SipD, SlrP, SopA, SopB/SigD, SopD, SopE, SopE2, SptP, SspH1, SteA, and SteB (6–8). Six of them have been shown to regulate actin cytoskeleton dynamics (reviewed in Ref. 9). 19 SPI-2 T3SS effectors have been identified: GogB, PipB, PipB2, SifA, SifB, SopD2, SseF, SlrP, SseG, SseI/SrfH, SseJ, SseK1, SseK2, SseL, SspH1, SspH2, SteA, SteB, and SteC. However, the biochemical functions of most of them remain unknown (reviewed in Ref. 10).The conventional paradigm, supported by in vivo and in vitro studies, is that the SPI-1-encoded T3SS is required for the invasion of M cells and cultured epithelial cells (11, 12) as well as for the inflammatory response of the intestinal cells, and that the SPI-2-encoded T3SS is essential for replication and survival within macrophages and the progression of a systemic infection (13). Recent evidence suggests that the boundaries between SPI-1 and SPI-2 function are not sharply defined: some SPI-1 effectors are detected hours or days after infection and SPI-2-encoded genes may be expressed before penetration of the intestinal epithelium. In addition, as can be noticed comparing the lists of effectors above, some effectors, including SlrP, can be secreted by both T3SS.SlrP (for Salmonella leucine-rich repeat protein) was identified as a S. enterica serovar Typhimurium host range factor by signature-tagged mutagenesis (14). A mutant in this gene has no difference in virulence with the wild-type strain when infecting calves but is 6-fold attenuated for mouse virulence after oral infection. This gene is located in a 2.9-kb DNA region with features of horizontal acquisition and has similarity to yopM from Yersinia spp. and ipaH from Shigella flexneri. The predicted protein contains 10 copies of a leucine-rich repeat signature, a protein motif frequently involved in protein-protein interactions. Other members of the leucine-rich repeat family in Salmonella are the effectors SspH1 and SspH2, which share 39 and 38% amino acid identity with SlrP, respectively. Similarity in the amino-terminal region of these three proteins helped to define a translocation signal that was also found in four other T3SS effectors: SifA, SifB, SseI, and SseJ (15). Although SlrP can be delivered into mammalian cells by both T3SS, its expression seems to be induced by RtsA, one of the main regulators of SPI-1, independently of HilA or InvF (16).Although the function of SlrP was completely unknown, the presence of leucine-rich repeats in this protein suggested that it may bind eukaryotic proteins during infection. In addition, recent reports have shown an enzymatic activity, E3 ubiquitin ligase, for effectors of the same family (17, 18).In this work we demonstrate that SlrP interacts with mammalian thioredoxin-1 (Trx). We also show that SlrP is an E3 ubiquitin ligase that can use Trx as a substrate. Our results support a model in which interaction of SlrP with Trx leads to a decrease in thioredoxin activity and triggers host cell death.     

Systematic analysis of the regulation of type three secreted effectors in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium

Background  

The type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions.