Identification of Clostridioides difficile mutants with increased daptomycin resistance.

Daptomycin is a cyclic lipopeptide antibiotic used to treat infections caused by some Gram-positive bacteria. Daptomycin disrupts synthesis of the peptidoglycan (PG) cell wall by inserting into the cytoplasmic membrane and binding multiple forms of the undecaprenyl carrier lipid required for PG synthesis. Membrane insertion requires phosphatidylglycerol, so studies of daptomycin can provide insight into assembly and maintenance of the cytoplasmic membrane. Here, we studied the effects of daptomycin on Clostridioides difficile, the leading cause of healthcare-associated diarrhea. We observed that growth of C. difficile strain R20291 in the presence of sub-MIC levels of daptomycin resulted in a chaining phenotype, minicell formation, and lysis-phenotypes broadly consistent with perturbation of membranes and PG synthesis. We also selected for and characterized eight mutants with elevated daptomycin resistance. The mutations in these mutants were mapped to four genes: cdsA (cdr20291_2041), ftsH2 (cdr20291_3396), esrR (cdr20291_1187), and draS (cdr20291_2456). Of these four genes, only draS has been characterized previously. Follow-up studies indicate these mutations confer daptomycin resistance by two general mechanisms: reducing the amount of phosphatidylglycerol in the cytoplasmic membrane (cdsA) or altering the regulation of membrane processes (ftsH2, esrR, and draS). Thus, the mutants described here provide insights into phospholipid synthesis and identify signal transduction systems involved in cell envelope biogenesis and stress response in C. difficile. IMPORTANCE: C. difficile is the leading cause of healthcare-associated diarrhea and is a threat to public health due to the risk of recurrent infections. Understanding biosynthesis of the atypical cell envelope of C. difficile may provide insight into novel drug targets to selectively inhibit C. difficile. Here, we identified mutations that increased daptomycin resistance and allowed us to better understand phospholipid synthesis, cell envelope biogenesis, and stress response in C. difficile.

Identification of the Extracytoplasmic Function σ Factor σP Regulon in Bacillus thuringiensis.

Bacillus thuringiensis and other members of the Bacillus cereus family are resistant to many ß-lactams. Resistance is dependent upon the extracytoplasmic function sigma factor σP. We used label-free quantitative proteomics to identify proteins whose expression was dependent upon σP. We compared the protein profiles of strains which either lacked σP or overexpressed σP. We identified 8 members of the σP regulon which included four ß-lactamases as well as three penicillin-binding proteins (PBPs). Using transcriptional reporters, we confirmed that these genes are induced by ß-lactams in a σP-dependent manner. These genes were deleted individually or in various combinations to determine their role in resistance to a subset of ß-lactams, including ampicillin, methicillin, cephalexin, and cephalothin. We found that different combinations of ß-lactamases and PBPs are involved in resistance to different ß-lactams. Our data show that B. thuringiensis utilizes a suite of enzymes to protect itself from ß-lactam antibiotics. IMPORTANCE Antimicrobial resistance is major concern for public health. ß-Lactams remain an important treatment option for many diseases. However, the spread of ß-lactam resistance continues to rise. Many pathogens acquire antibiotic resistance from environmental bacteria. Thus, understanding ß-lactam resistance in environmental strains may provide insights into additional mechanisms of antibiotic resistance. Here, we describe how a single regulatory system, σP, in B. thuringiensis controls expression of multiple genes involved in resistance to ß-lactams. Our findings indicate that some of these genes are partially redundant. Our data also suggest that the large number of genes controlled by σP results in increased resistance to a wider range of ß-lactam classes than any single gene could provide.

Activation of the extracytoplasmic function σ factor σV by lysozyme in Clostridioides difficile.

Clostridioides difficile is naturally resistant to high levels of lysozyme an important component of the innate immune defense system. C. difficile encodes both constitutive as well as inducible lysozyme resistance genes. The inducible lysozyme resistance genes are controlled by an alternative σ factor σV that belongs to the Extracytoplasmic function σ factor family. In the absence of lysozyme, the activity of σV is inhibited by the anti-σ factor RsiV. In the presence of lysozyme RsiV is destroyed via a proteolytic cascade that leads to σV activation and increased lysozyme resistance. This review highlights how activity of σV is controlled.

The Penicillin-Binding Protein PbpP Is a Sensor of ß-Lactams and Is Required for Activation of the Extracytoplasmic Function σ Factor σP in Bacillus thuringiensis.

ß-Lactams are a class of antibiotics that target the synthesis of peptidoglycan, an essential component of the cell wall. ß-Lactams inhibit the function of penicillin-binding proteins (PBPs), which form the cross-links between strands of peptidoglycan. Resistance to ß-lactams complicates the treatment of bacterial infections. In recent years, the spread of ß-lactam resistance has increased with growing intensity. Resistance is often conferred by ß-lactamases, which inactivate ß-lactams, or the expression of alternative ß-lactam-resistant PBPs. σP is an extracytoplasmic function (ECF) σ factor that controls ß-lactam resistance in the species Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis σP is normally held inactive by the anti-σ factor RsiP. σP is activated by ß-lactams that trigger the proteolytic destruction of RsiP. Here, we identify the penicillin-binding protein PbpP and demonstrate its essential role in the activation of σP Our data show that PbpP is required for σP activation and RsiP degradation. Our data suggest that PbpP acts as a ß-lactam sensor since the binding of a subset of ß-lactams to PbpP is required for σP activation. We find that PbpP likely directly or indirectly controls site 1 cleavage of RsiP, which results in the degradation of RsiP and, thus, σP activation. σP activation results in increased expression of ß-lactamases and, thus, increased ß-lactam resistance. This work is the first report of a PBP acting as a sensor for ß-lactams and controlling the activation of an ECF σ factor.IMPORTANCE The bacterial cell envelope is the target for numerous antibiotics. Many antibiotics target the synthesis of peptidoglycan, which is a central metabolic pathway essential for bacterial survival. One of the most important classes of antibiotics has been ß-lactams, which inhibit the transpeptidase activity of penicillin-binding proteins to decrease the cross-linking of peptidoglycan and the strength of the cell wall. While ß-lactam antibiotics have historically proven to be effective, resistance to ß-lactams is a growing problem. The ECF σ factor σP is required for ß-lactam resistance in B. thuringiensis and close relatives, including B. anthracis Here, we provide insight into the mechanism of activation of σP by ß-lactams.

Signal Peptidase-Mediated Cleavage of the Anti-σ Factor RsiP at Site 1 Controls σP Activation and ß-Lactam Resistance in Bacillus thuringiensis.

In Bacillus thuringiensis, ß-lactam antibiotic resistance is controlled by the extracytoplasmic function (ECF) σ factor σP. σP activity is inhibited by the anti-σ factor RsiP. In the presence of ß-lactam antibiotics, RsiP is degraded and σP is activated. Previous work found that RsiP degradation requires cleavage of RsiP at site 1 by an unknown protease, followed by cleavage at site 2 by the site 2 protease RasP. The penicillin-binding protein PbpP acts as a sensor for ß-lactams. PbpP initiates σP activation and is required for site 1 cleavage of RsiP but is not the site 1 protease. Here, we describe the identification of a signal peptidase, SipP, which cleaves RsiP at a site 1 signal peptidase cleavage site and is required for σP activation. Finally, many B. anthracis strains are sensitive to ß-lactams yet encode the σP-RsiP signal transduction system. We identified a naturally occurring mutation in the signal peptidase cleavage site of B. anthracis RsiP that renders it resistant to SipP cleavage. We find that B. anthracis RsiP is not degraded in the presence of ß-lactams. Altering the B. anthracis RsiP site 1 cleavage site by a single residue to resemble B. thuringiensis RsiP results in ß-lactam-dependent degradation of RsiP. We show that mutation of the B. thuringiensis RsiP cleavage site to resemble the sequence of B. anthracis RsiP blocks degradation by SipP. The change in the cleavage site likely explains many reasons why B. anthracis strains are sensitive to ß-lactams. IMPORTANCE ß-Lactam antibiotics are important for the treatment of many bacterial infections. However, resistance mechanisms have become increasingly more prevalent. Understanding how ß-lactam resistance is conferred and how bacteria control expression of ß-lactam resistance is important for informing the future treatment of bacterial infections. σP is an alternative σ factor that controls the transcription of genes that confer ß-lactam resistance in Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis. Here, we identify a signal peptidase as the protease required for initiating activation of σP by the degradation of the anti-σ factor RsiP. The discovery that the signal peptidase SipP is required for σP activation highlights an increasing role for signal peptidases in signal transduction, as well as in antibiotic resistance.

Activation of the Extracytoplasmic Function σ Factor σP by ß-Lactams in Bacillus thuringiensis Requires the Site-2 Protease RasP.

Bacteria can utilize alternative σ factors to regulate sets of genes in response to changes in the environment. The largest and most diverse group of alternative σ factors are the extracytoplasmic function (ECF) σ factors. σP is an ECF σ factor found in Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Previous work showed that σP is induced by ampicillin, a ß-lactam antibiotic, and required for resistance to ampicillin. However, it was not known how activation of σP is controlled or what other antibiotics may activate σP Here, we report that activation of σP is specific to a subset of ß-lactams and that σP is required for resistance to these ß-lactams. We demonstrate that activation of σP is controlled by the proteolytic destruction of the anti-σ factor RsiP and that degradation of RsiP requires multiple proteases. Upon exposure to ß-lactams, the extracellular domain of RsiP is cleaved by an unknown protease, which we predict cleaves at site-1. Following cleavage by the unknown protease, the N terminus of RsiP is further degraded by the site-2 intramembrane protease RasP. Our data indicate that RasP cleavage of RsiP is not the rate-limiting step in σP activation. This proteolytic cascade leads to activation of σP, which induces resistance to ß-lactams likely via increased expression of ß-lactamases.IMPORTANCE The discovery of antibiotics to treat bacterial infections has had a dramatic and positive impact on human health. However, shortly after the introduction of a new antibiotic, bacteria often develop resistance. The bacterial cell envelope is essential for cell viability and is the target of many of the most commonly used antibiotics, including ß-lactam antibiotics. Resistance to ß-lactams is often dependent upon ß-lactamases. In B. cereus, B. thuringiensis, and some B. anthracis strains, the expression of some ß-lactamases is inducible. This inducible ß-lactamase expression is controlled by activation of an alternative σ factor called σP Here, we show that ß-lactam antibiotics induce σP activation by degradation of the anti-σ factor RsiP.

Activation of the extracytoplasmic function σ factor σV by lysozyme.

σV is an extracytoplasmic function (ECF) σ factor that is found exclusively in Firmicutes including Bacillus subtilis and the opportunistic pathogens Clostridioides difficile and Enterococcus faecalis. σV is activated by lysozyme and is required for lysozyme resistance. The activity of σV is normally inhibited by the anti-σ factor RsiV, a transmembrane protein. RsiV acts as a receptor for lysozyme. The binding of lysozyme to RsiV triggers a signal transduction cascade which results in degradation of RsiV and activation of σV . Like the anti-σ factors for several other ECF σ factors, RsiV is degraded by a multistep proteolytic cascade that is regulated at the step of site-1 cleavage. Unlike other anti-σ factors, site-1 cleavage of RsiV is not dependent upon a site-1 protease whose activity is regulated. Instead constitutively active signal peptidase cleaves RsiV at site-1 in a lysozyme-dependent manner. The activation of σV leads to the transcription of genes, which encode proteins required for lysozyme resistance.

Glycerol Monolaurate (GML) and a Nonaqueous Five-Percent GML Gel Kill Bacillus and Clostridium Spores.

Glycerol monolaurate is a broadly antimicrobial fatty acid monoester, killing bacteria, fungi, and enveloped viruses. The compound kills stationary-phase cultures of Bacillus anthracis, suggesting that the molecule may kill spores. In this study, we examined the ability of glycerol monolaurate alone or solubilized in a nonaqueous gel to kill vegetative cells and spores of aerobic B. anthracis, B. subtilis, and B. cereus and anaerobic Clostridium perfringens and Clostridium (Clostridioides) difficile. Glycerol monolaurate alone was bactericidal for all five organisms tested. Glycerol monolaurate alone was effective in killing spores. When solubilized in a nonaqueous gel, the glycerol monolaurate gel was bactericidal for all spores tested. The data suggest that glycerol monolaurate nonaqueous gel could be effective in decontaminating environmental and body surfaces, such as skin.IMPORTANCEBacillus and Clostridium spores are known to be highly resistant to killing, persisting on environmental and human body surfaces for long periods of time. In favorable environments, these spores may germinate and cause human diseases. It is thus important to identify agents that can be used on both environmental and human skin and mucosal surfaces and that are effective in killing spores. We previously showed that the fatty acid monoester glycerol monolaurate (GML) kills stationary-phase cultures of Bacillus anthracis Since such cultures are likely to contain spores, it is possible that GML and a human-use-approved GML nonaqueous gel would kill Bacillus and Clostridium spores. The significance of our studies is that we have identified GML, and, to a greater extent, GML solubilized in a nonaqueous gel, as effective in killing spores from both bacterial genera.

Ferric Uptake Regulator Fur Control of Putative Iron Acquisition Systems in Clostridium difficile.

UNLABELLED: Clostridium difficile is an anaerobic, Gram-positive, spore-forming opportunistic pathogen and is the most common cause of hospital-acquired infectious diarrhea. Although iron acquisition in the host is a key to survival of bacterial pathogens, high levels of intracellular iron can increase oxidative damage. Therefore, expression of iron acquisition mechanisms is tightly controlled by transcriptional regulators. We identified a C. difficile homologue of the master bacterial iron regulator Fur. Using targetron mutagenesis, we generated a fur insertion mutant of C. difficile. To identify the genes regulated by Fur in C. difficile, we used microarray analysis to compare transcriptional differences between the fur mutant and the wild type when grown in high-iron medium. The fur mutant had increased expression of greater than 70 transcriptional units. Using quantitative reverse transcriptase PCR (qRT-PCR), we analyzed several of the Fur-regulated genes identified by the microarray and verified that they are both iron and Fur regulated in C. difficile. Among those Fur- and iron-repressed genes were C. difficile genes encoding 7 putative cation transport systems of different classes. We found that Fur was able to bind the DNA upstream of three Fur-repressed genes in electrophoretic mobility shift assays. We also demonstrate that expression of Fur-regulated putative iron acquisition systems was increased during C. difficile infection using the hamster model. Our data suggest that C. difficile expresses multiple iron transport mechanisms in response iron depletion in vitro and in vivo. IMPORTANCE: Clostridium difficile is the most common cause of hospital-acquired infectious diarrhea and has been recently classified as an "urgent" antibiotic resistance threat by the CDC. To survive and cause disease, most bacterial pathogens must acquire the essential enzymatic cofactor iron. While import of adequate iron is essential for most bacterial growth, excess intracellular iron can lead to extensive oxidative damage. Thus, bacteria must regulate iron import to maintain iron homeostasis. We demonstrate here that C. difficile regulates expression of several putative iron acquisition systems using the transcriptional regulator Fur. These import mechanisms are induced under iron-limiting conditions in vitro and during C. difficile infection of the host. This suggests that during a C. difficile infection, iron availability is limited in vivo.

Clostridium difficile extracytoplasmic function σ factor σV regulates lysozyme resistance and is necessary for pathogenesis in the hamster model of infection.

Clostridium difficile is a clinically important pathogen and the most common cause of hospital-acquired infectious diarrhea. Expression of the C. difficile gene csfV, which encodes σ(V), an extracytoplasmic function σ factor, is induced by lysozyme, which damages the peptidoglycan of bacteria. Here we show that σ(V) is required for lysozyme resistance in C. difficile. Using microarray analysis, we identified the C. difficile genes whose expression is dependent upon σ(V) and is induced by lysozyme. Although the peptidoglycan of wild-type C. difficile is intrinsically highly deacetylated, we have found that exposure to lysozyme leads to additional peptidoglycan deacetylation. This lysozyme-induced deacetylation is dependent upon σ(V). Expression of pdaV, which encodes a putative peptidoglycan deacetylase, was able to increase lysozyme resistance of a csfV mutant. The csfV mutant strain is severely attenuated compared to wild-type C. difficile in a hamster model of C. difficile-associated disease. We conclude that the σ(V) signal transduction system, which senses the host innate immune defense enzyme lysozyme, is required for lysozyme resistance and is necessary during C. difficile infection.

Production of the cannibalism toxin SDP is a multistep process that requires SdpA and SdpB.

During the early stages of sporulation, a subpopulation of Bacillus subtilis cells secrete toxins that kill their genetically identical siblings in a process termed cannibalism. One of these toxins is encoded by the sdpC gene of the sdpABC operon. The active form of the SDP toxin is a 42-amino-acid peptide with a disulfide bond which is processed from an internal fragment of pro-SdpC. The factors required for the processing of pro-SdpC into mature SDP are not known. We provide evidence that pro-SdpC is secreted via the general secretory pathway and that signal peptide cleavage is a required step in the production of SDP. We also demonstrate that SdpAB are essential to produce mature SDP, which has toxin activity. Our data indicate that SdpAB are not required for secretion, translation, or stability of SdpC. Thus, SdpAB may participate in a posttranslation step in the production of SDP. The mature form of the SDP toxin contains a disulfide bond. Our data indicate that while the disulfide bond does increase activity of SDP, it is not essential for SDP activity. We demonstrate that the disulfide bond is formed independently of SdpAB. Taken together, our data suggest that SDP production is a multistep process and that SdpAB are required for SDP production likely by controlling, directly or indirectly, cleavage of SDP from the pro-SdpC precursor.

Extra cytoplasmic function σ factor activation.

The bacterial cell envelope is essential for cell viability and is a target for numerous antibiotics and host immune defenses. Thus bacteria must sense and respond to damage to the cell envelope. Many bacteria utilize alternative σ factors such as extracytoplasmic function (ECF) σ factors to respond to cell envelope stress. Although ECF σ factors are utilized by both Gram negative and Gram positive bacteria to respond to cell envelope stress, the mechanisms of sensing differ. In this review, we examine the events and proteins that are required for activation of two model extracytoplasmic function σ factors, σ(E) in E. coli and σ(W) in B. subtilis.

The Bacillus subtilis extracytoplasmic function σ factor σ(V) is induced by lysozyme and provides resistance to lysozyme.

Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative σ factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative σ factors are the extracytoplasmic function (ECF) σ factors. In this paper, we demonstrate that the expression of the ECF σ factor σ(V) in Bacillus subtilis is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in sigV results in increased sensitivity to lysozyme killing, suggesting that σ(V) is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene yrhL (here referred to as oatA for O-acetyltransferase) is in a four-gene operon which includes sigV and rsiV. In quantitative RT-PCR experiments, the expression of oatA is induced by lysozyme stress. Lysozyme induction of oatA is dependent upon σ(V). Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for σ(V)-dependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF σ factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF σ factors contributed to lysozyme resistance. We found that sigX and sigM mutations alone had very little effect on lysozyme resistance but when combined with a sigV mutation resulted in significantly greater lysozyme sensitivity than the sigV mutation alone. This suggests that sigV, sigX, and sigM may act synergistically to control lysozyme resistance. In addition, we show that two ECF σ factor-regulated genes, dltA and pbpX, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which B. subtilis utilizes to avoid killing by lysozyme.

PrsW is required for colonization, resistance to antimicrobial peptides, and expression of extracytoplasmic function σ factors in Clostridium difficile.

Clostridium difficile is an anaerobic, Gram-positive, spore-forming, opportunistic pathogen that is the most common cause of hospital-acquired infectious diarrhea. In numerous pathogens, stress response mechanisms are required for survival within the host. Extracytoplasmic function (ECF) σ factors are a major family of signal transduction systems, which sense and respond to extracellular stresses. We have identified three C. difficile ECF σ factors. These ECF σ factors, CsfT, CsfU, and CsfV, induce their own expressions and are negatively regulated by their cognate anti-σ factors, RsiT, RsiU, and RsiV, respectively. The levels of expression of these ECF σ factors increase following exposure to the antimicrobial peptides bacitracin and/or lysozyme. The expressions of many ECF σ factors are controlled by site 1 and site 2 proteases, which cleave anti-σ factors. Using a retargeted group II intron, we generated a C. difficile mutation in prsW, a putative site 1 protease. The C. difficile prsW mutant exhibited decreased levels of expression of CsfT and CsfU but not of CsfV. When expressed in a heterologous host, C. difficile PrsW was able to induce the degradation of RsiT but not of RsiU. When the prsW mutant was tested in competition assays against its isogenic parent in the hamster model of C. difficile infection, we found that the prsW mutant was 30-fold less virulent than the wild type. The prsW mutant was also significantly more sensitive to bacitracin and lysozyme than the wild type in in vitro competition assays. Taken together, these data suggest that PrsW likely regulates the activation of the ECF σ factor CsfT in C. difficile and controls the resistance of C. difficile to antimicrobial peptides that are important for survival in the host.

Type 2 secretion promotes enterohemorrhagic Escherichia coli adherence and intestinal colonization.

Enterohemorrhagic Escherichia coli (EHEC) is a noninvasive food-borne pathogen that colonizes the distal ileum and colon. Proteins encoded in the EHEC locus of enterocyte effacement (LEE) pathogenicity island are known to contribute to this pathogen's adherence to epithelial cells and intestinal colonization. The role of non-LEE-encoded proteins in these processes is not as clear. We found that the Z2053 gene (designated adfO here), a gene located in a cryptic EHEC prophage, exhibits similarity to adherence and/or colonization factor genes found in several other enteric pathogens. An EHEC adfO mutant exhibited marked reductions in adherence to HeLa cells and in the secretion of several proteins into the supernatant. YodA, one of these secreted proteins, was found to be a substrate of the EHEC pO157-encoded type 2 secretion system (T2SS). Both the T2SS and YodA proved to be essential for EHEC adherence to cultured HeLa cell monolayers. Using an infant rabbit model of infection, we found that the adfO mutation did not affect colonization but that the colonization of an etpC (T2SS) mutant was reduced approximately 5-fold. A strain deficient in YodA had a more severe colonization defect; however, this strain also exhibited a growth defect in vitro. Overall, our findings indicate that the pO157-encoded T2SS contributes to EHEC adherence and intestinal colonization and thus show that EHEC pathogenicity depends on type 2 secretion as well as type 3 secretion.

The Salmonella enterica serovar typhimurium-encoded type III secretion systems can translocate Chlamydia trachomatis proteins into the cytosol of host cells.

Chlamydia trachomatis is an obligate, intracellular pathogen that is a major cause of preventable blindness and infertility worldwide. Although the published genome sequence suggests that C. trachomatis encodes a type III secretion system, the lack of genetic tools for studying Chlamydia has hindered the examination of this potentially important class of virulence genes. We have developed a technique to identify Chlamydia proteins that can be translocated into the host cell cytoplasm by a type III secretion system. We have selected several Chlamydia proteins and tagged them with a multiple peptide motif element called F8M4. Epitopes contained in the F8M4 tag allow us to use tools corresponding to different arms of the adaptive immune system to detect the expression and translocation of these proteins by Salmonella enterica serovar Typhimurium. In particular, CD8(+)-T-cell reactivity can be used to detect the translocation of F8M4-tagged proteins into the cytoplasm of host cells. We have found that CD8(+)-T-cell activity assays are sensitive enough to detect translocation of even a small amount of F8M4-tagged protein. We have used CD8(+)-T-cell activity to show that CopN, a Chlamydia protein previously shown to be translocated by Yersinia type III secretion, can be translocated by the Salmonella pathogenicity island 1 (SPI-1) type III secretion system. Additionally, we demonstrate that CopD and Pkn5, two Chlamydia proteins hypothesized to be substrates of a type III secretion system, are translocated via the SPI-2 type III secretion system of serovar Typhimurium. The epitope tag system described here can be used more generally to examine the expression and subcellular compartmentalization of bacterial proteins deployed during the interaction of pathogens with mammalian cells.

Identification of GtgE, a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium.

The Gifsy-2 temperate bacteriophage of Salmonella enterica serovar Typhimurium contributes significantly to the pathogenicity of strains that carry it as a prophage. Previous studies have shown that Gifsy-2 encodes SodCI, a periplasmic Cu/Zn superoxide dismutase, and at least one additional virulence factor. Gifsy-2 encodes a Salmonella pathogenicity island 2 type III secreted effector protein. Sequence analysis of the Gifsy-2 genome also identifies several open reading frames with homology to those of known virulence genes. However, we found that null mutations in these genes did not individually have a significant effect on the ability of S. enterica serovar Typhimurium to establish a systemic infection in mice. Using deletion analysis, we have identified a gene, gtgE, which is necessary for the full virulence of S. enterica serovar Typhimurium Gifsy-2 lysogens. Together, GtgE and SodCI account for the contribution of Gifsy-2 to S. enterica serovar Typhimurium virulence in the murine model.