Lydia M. Bogomolnaya

'Form variation' of the O12 antigen is critical for persistence of Salmonella Typhimurium in the murine intestine

Salmonella enterica subspecies I serotypes are responsible for the vast majority of salmonellosis in mammals and birds, yet only a few factors specific to this group that allow them to persist in this niche have been identified. We show that STM0557, a S. enterica subspecies I‐specific gene encoding an inner membrane protein, is critical for faecal shedding and intestinal persistence of S. enterica serotype Typhimurium ATCC14028 in Salmonella‐resistant mice, but mutations in this gene do not diminish short‐term intestinal colonization or invasion of cultured epithelial cells. STM0557 and two neighbouring genes, located on a pathogenicity island termed SPI‐16, resemble genes of the gtrA,B, gtr(type) cluster in seroconverting bacteriophages. In general, the gtr genes encode proteins responsible for serotype conversion of the infected bacterium by addition glucose residues to repeating O‐antigen subunits of lipopolysaccharide (LPS). In lysogenized Shigella, such modifications have been previously shown to be constitutively expressed and to facilitate invasion of host cells. We show that serotype Typhimurium gtr orthologues, STM0557–0559, are responsible for ‘form variation’ or glucosylation of the O12 antigen galactose (4 position) to generate the 12‐2 variant. Form variation in Typhimurium is not constitutive, but occurred upon exposure and during intracellular growth of serotype Typhimurium in J774 macrophages. Our data suggest that the 12‐2 antigen is a S. enterica subspecies I‐specific LPS modification that enhances long‐term intestinal colonization, and is in contrast to the role of O‐antigen variation described for Shigella.

Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium.

We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

The ABC-Type Efflux Pump MacAB Protects Salmonella enterica serovar Typhimurium from Oxidative Stress

ABSTRACT Multidrug efflux pumps are integral membrane proteins known to actively excrete antibiotics. The macrolide-specific pump MacAB, the only ABC-type drug efflux pump in Salmonella, has previously been linked to virulence in mice. The molecular mechanism of this link between macAB and infection is unclear. We demonstrate that macAB plays a role in the detoxification of reactive oxygen species (ROS), compounds that salmonellae are exposed to at various stages of infection. macAB is induced upon exposure to H2O2 and is critical for survival of Salmonella enterica serovar Typhimurium in the presence of peroxide. Furthermore, we determined that macAB is required for intracellular replication inside J774.A1 murine macrophages but is not required for survival in ROS-deficient J774.D9 macrophages. macAB mutants also had reduced survival in the intestine in the mouse colitis model, a model characterized by a strong neutrophilic intestinal infiltrate where bacteria may experience the cytotoxic actions of ROS. Using an Amplex red-coupled assay, macAB mutants appear to be unable to induce protection against exogenous H2O2 in vitro, in contrast to the isogenic wild type. In mixed cultures, the presence of the wild-type organism, or media preconditioned by the growth of the wild-type organism, was sufficient to rescue the macAB mutant from peroxide-mediated killing. Our data indicate that the MacAB drug efflux pump has functions beyond resistance to antibiotics and plays a role in the protection of Salmonella against oxidative stress. Intriguingly, our data also suggest the presence of a soluble anti-H2O2 compound secreted by Salmonella cells through a MacAB-dependent mechanism. IMPORTANCE The ABC-type multidrug efflux pump MacAB is known to be required for Salmonella enterica serovar Typhimurium virulence after oral infection in mice, yet the function of this pump during infection is unknown. We show that this pump is necessary for colonization of niches in infected mice where salmonellae encounter oxidative stress during infection. MacAB is required for growth in cultured macrophages that produce reactive oxygen species (ROS) but is not needed in macrophages that do not generate ROS. In addition, we show that MacAB is required to resist peroxide-mediated killing in vitro and for the inactivation of peroxide in the media. Finally, wild-type organisms, or supernatant from wild-type organisms grown in the presence of peroxide, rescue the growth defect of macAB mutants in H2O2. MacAB appears to participate in the excretion of a compound that induces protection against ROS-mediated killing, revealing a new role for this multidrug efflux pump. The ABC-type multidrug efflux pump MacAB is known to be required for Salmonella enterica serovar Typhimurium virulence after oral infection in mice, yet the function of this pump during infection is unknown. We show that this pump is necessary for colonization of niches in infected mice where salmonellae encounter oxidative stress during infection. MacAB is required for growth in cultured macrophages that produce reactive oxygen species (ROS) but is not needed in macrophages that do not generate ROS. In addition, we show that MacAB is required to resist peroxide-mediated killing in vitro and for the inactivation of peroxide in the media. Finally, wild-type organisms, or supernatant from wild-type organisms grown in the presence of peroxide, rescue the growth defect of macAB mutants in H2O2. MacAB appears to participate in the excretion of a compound that induces protection against ROS-mediated killing, revealing a new role for this multidrug efflux pump.

An Increase in Mitochondrial DNA Promotes Nuclear DNA Replication in Yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD+-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.

A comparison of cecal colonization of Salmonella enterica serotype Typhimurium in white leghorn chicks and Salmonella-resistant mice.

BackgroundSalmonellosis is one of the most important bacterial food borne illnesses worldwide. A major source of infection for humans is consumption of chicken or egg products that have been contaminated with Salmonella enterica serotype Typhimurium, however our knowledge regarding colonization and persistence factors in the chicken is small.ResultsWe compared intestinal and systemic colonization of 1-week-old White Leghorn chicks and Salmonella-resistant CBA/J mice during infection with Salmonella enterica serotype Typhimurium ATCC14028, one of the most commonly studied isolates. We also studied the distribution of wild type serotype Typhimurium ATCC14028 and an isogenic invA mutant during competitive infection in the cecum of 1-week-old White Leghorn chicks and 8-week-old CBA/J mice. We found that although the systemic levels of serotype Typhimurium in both infected animal models are low, infected mice have significant splenomegaly beginning at 15 days post infection. In the intestinal tract itself, the cecal contents are the major site for recovery of serotype Typhimurium in the cecum of 1-week-old chicks and Salmonella-resistant mice. Additionally we show that only a small minority of Salmonellae are intracellular in the cecal epithelium of both infected animal models, and while SPI-1 is important for successful infection in the murine model, it is important for association with the cecal epithelium of 1-week-old chicks. Finally, we show that in chicks infected with serotype Typhimurium at 1 week of age, the level of fecal shedding of this organism does not reflect the level of cecal colonization as it does in murine models.ConclusionIn our study, we highlight important differences in systemic and intestinal colonization levels between chick and murine serotype Typhimurium infections, and provide evidence that suggests that the role of SPI-1 may not be the same during colonization of both animal models.

Abrogation of the Twin Arginine Transport System in Salmonella enterica Serovar Typhimurium Leads to Colonization Defects during Infection

TatC (STM3975) is a highly conserved component of the Twin Arginine Transport (Tat) systems that is required for transport of folded proteins across the inner membrane in gram-negative bacteria. We previously identified a ΔtatC mutant as defective in competitive infections with wild type ATCC14028 during systemic infection of Salmonella-susceptible BALB/c mice. Here we confirm these results and show that the ΔtatC mutant is internalized poorly by cultured J774-A.1 mouse macrophages a phenotype that may be related to the systemic infection defect. This mutant is also defective for short-term intestinal and systemic colonization after oral infection of BALB/c mice and is shed in reduced numbers in feces from orally infected Salmonella-resistant (CBA/J) mice. We show that the ΔtatC mutant is highly sensitive to bile acids perhaps resulting in the defect in intestinal infection that we observe. Finally, the ΔtatC mutant has an unusual combination of motility phenotypes in Salmonella; it is severely defective for swimming motility but is able to swarm well. The ΔtatC mutant has a lower amount of flagellin on the bacterial surface during swimming motility but normal levels under swarming conditions.

Identification of novel factors involved in modulating motility of Salmonella enterica serotype typhimurium.

Salmonella enterica serotype Typhimurium can move through liquid using swimming motility, and across a surface by swarming motility. We generated a library of targeted deletion mutants in Salmonella Typhimurium strain ATCC14028, primarily in genes specific to Salmonella, that we have previously described. In the work presented here, we screened each individual mutant from this library for the ability to move away from the site of inoculation on swimming and swarming motility agar. Mutants in genes previously described as important for motility, such as flgF, motA, cheY are do not move away from the site of inoculation on plates in our screens, validating our approach. Mutants in 130 genes, not previously known to be involved in motility, had altered movement of at least one type, 9 mutants were severely impaired for both types of motility, while 33 mutants appeared defective on swimming motility plates but not swarming motility plates, and 49 mutants had reduced ability to move on swarming agar but not swimming agar. Finally, 39 mutants were determined to be hypermotile in at least one of the types of motility tested. Both mutants that appeared non-motile and hypermotile on plates were assayed for expression levels of FliC and FljB on the bacterial surface and many of them had altered levels of these proteins. The phenotypes we report are the first phenotypes ever assigned to 74 of these open reading frames, as they are annotated as ‘hypothetical genes’ in the Typhimurium genome.

Subspecies IIIa and IIIb Salmonellae Are Defective for Colonization of Murine Models of Salmonellosis Compared to Salmonella enterica subsp. I Serovar Typhimurium

Non-subspecies I salmonellae are commensals of cold-blooded vertebrates and cause sporadic disease in mammals. The reasons why non-subspecies I salmonellae do not circulate in populations of warm-blooded vertebrates, but instead only cause occasional disease in this niche, are unknown. We examined the ability of Salmonella enterica subsp. IIIa (subsp. arizonae) and subsp. IIIb (subsp. diarizonae) isolates to grow competitively with subspecies I (serovar Typhimurium) ATCC 14028 in vitro, to colonize Salmonella-sensitive BALB/c mice, and to persist in the intestine of Salmonella-resistant CBA/J mice in competitive infections. Subspecies IIIa had severely reduced intestinal colonization, intestinal persistence, and systemic spread in mice. Subspecies IIIa is nonmotile on swarming agar and thus may also have reduced motility under viscous conditions in vivo. Surprisingly, subspecies IIIb colonizes the intestinal tract of BALB/c mice normally yet does not spread systemically. Subspecies IIIb colonization of the intestine of CBA/J mice is reduced late in infection. In order to understand why these isolates do not colonize systemic sites, we determined that subspecies IIIa and IIIb are not internalized well and do not replicate in J774-A.1 murine macrophages, despite normal adherence to these cells. We further show that selected effectors of both type III secretion systems 1 and 2 are secreted by subspecies IIIa and IIIb in vitro but that each of these isolates secretes a different combination of effectors. We outline the phenotypic differences between these subspecies and subspecies I and provide a possible explanation for the inability of these strains to spread systemically in murine models.

A new enrichment approach identifies genes that alter cell cycle progression in Saccharomyces cerevisiae

Mechanisms that coordinate cell growth with division are thought to determine the timing of initiation of cell division and to limit overall cell proliferation. To identify genes involved in this process in Saccharomyces cerevisiae, we describe a method that does not rely on cell size alterations or resistance to pheromone. Instead, our approach was based on the cell surface deposition of the Flo1p protein in cells having passed START. We found that over-expression of HXT11 (which encodes a plasma membrane transporter), PPE1 (coding for a protein methyl esterase), or SIK1 (which encodes a protein involved in rRNA processing) shortened the duration of the G1 phase of the cell cycle, prior to the initiation of DNA replication. In addition, we found that, although SIK1 was not part of a mitotic checkpoint, SIK1 over-expression caused spindle orientation defects and sensitized G2/M checkpoint mutant cells. Thus, unlike HXT11 and PPE1, SIK1 over-expression is also associated with mitotic functions. Overall, we used a novel enrichment approach and identified genes that were not previously associated with cell cycle progression. This approach can be extended to other organisms.

Roles of the RAM signaling network in cell cycle progression in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Hym1p, Mob2p, Tao3p, Cbk1p, Sog2p and Kic1p proteins are thought to function together in the RAM signaling network, which controls polarized growth, cell separation and cell integrity. Whether these proteins also function as a network to affect cell proliferation is not clear. Here we examined cells lacking or over-expressing RAM components, and evaluated the timing of initiation of DNA replication in each case. Our results suggest opposing roles of RAM proteins, where only Hym1p can promote the transition from the G1 to S phase of the cell cycle. We also uncovered additive growth defects in strains lacking several pair-wise combinations of RAM proteins, possibly arguing for multiple roles of RAM components in the overall control of cell proliferation. Finally, our findings suggest that Hym1p requires the Dcr2p phosphatase to promote the G1/S transition, but it does not require the G1 cyclin Cln3p or the RAS pathway. Taken together, our results point to a complex regulation of cell proliferation by RAM proteins, in a non-uniform manner that was not previously anticipated.