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V L Miller

Publications and source records attributed to V L Miller.

At least 55 records · Page 3Linked to original sources
PubMed1996

A bifunctional urease enhances survival of pathogenic Yersinia enterocolitica and Morganella morganii at low pH.

To infect a susceptible host, the gastrointestinal pathogen Yersinia enterocolitica must survive passage through the acid environment of the stomach. In this study, we showed that Y. enterocolitica serotype O8 survives buffered acidic conditions as low as pH 1.5 for long periods of time provided urea is available. Acid tolerance required an unusual cytoplasmically located urease that was activated 780-fold by low-pH conditions. Acid tolerance of Helicobacter species has also been attributed to urease activity, but in that case urease was not specifically activated by low-pH conditions. A ure mutant strain of Y. enterocolitica was constructed which was hypersensitive to acidic conditions when urea was available and, unlike the parental strain, was unable to grow when urea was the sole nitrogen source. Examination of other urease-producing gram-negative bacteria indicated that Morganella morganii survives in acidic conditions but Escherichia coli 1021, Klebsiella pneumoniae, Proteus mirabilis, Providencia stuartii, and Pseudomonas aeruginosa do not. Consistent with these results, biochemical evidence demonstrated that Y. enterocolitica and M. morganii ureases were activated in vitro by low pH with an unusually low activity optimum of pH 5.5. In whole cells activation occurred as medium values decreased below pH 3.0 for Y. enterocolitica and pH 5.5 for M. morganii, suggesting that in vivo activation occurs as a result of cytoplasmic acidification. DNA sequence analysis of portions of the M. morganii ure locus showed that the predicted primary structure of the enzyme structural subunits is most similar to those of Y. enterocolitica urease. One region of similarity between these two ureases located near the active site is distinct from most other ureases but is present in the urease of Lactobacillus fermentum. This region of similarity may be responsible for the unique properties of the Y. enterocolitica and M. morganii ureases since the L. fermentum urease also has been shown to have a low pH optimum for activity.

Acids↗
PubMed1995

Tissue-culture invasion: fact or artefact?

Although widely used, tissue-culture assays cannot be exact models of the conditions that are met in vivo by pathogenic bacteria. However, recent studies of specific mutants suggest that the model is good for highly invasive bacteria, but it remains to be seen if this is true for weakly invasive bacteria.

Animals↗
PubMed1995

Salmonella enteritidis has a homologue of tolC that is required for virulence in BALB/c mice.

The ability of Salmonella to invade tissue culture cells is correlated with virulence. Therefore, the tissue culture invasion model has been used extensively to study this process and to identify the bacterial genes involved and their products. Described here is the further characterization of a Salmonella enteritidis mutant (SM6T) originally identified as a non-invasive for tissue culture cells. A chromosomal DNA fragment complementing this defect was cloned and sequenced. The derived protein sequence is 89% identical to TolC from Escherichia coli, an outer membrane protein required for the signal peptide-independent transport of alpha-haemolysin and colicin V. Therefore, sinA was renamed tolC and is referred to in this text as tolCs to distinguish it from tolC of E. coli. TolCs and TolC are functionally similar since tolC can complement the invasion-defective phenotype of a tolCs mutant, and tolCs is required for export of alpha-haemolysin by Salmonella. The tolCs mutant is avirulent for mice when administered by the oral route, suggesting that the gene is important for virulence. Further characterization of the tolCs mutant indicated that like tolC mutants it is more sensitive than the wild-type strain to various detergents, antibiotics and dyes. This mutant is more sensitive to Triton X-100 only when associated with the monolayer, and the invasion-defective phenotype appears to be artifact of this sensitivity. In addition, the tolCs mutant is more sensitive to the bactericidal activity of human serum. Therefore, the avirulent phenotype could be the result of an inability to secrete a necessary virulence factor, or an increased sensitivity to complement and detergents as a result of a subtle alteration in the lipopolysaccharide (LPS) associated with tolC mutations.

Amino Acid Sequence↗
PubMed1995

Pathogenesis of defined invasion mutants of Yersinia enterocolitica in a BALB/c mouse model of infection.

It has been hypothesized for many years that the ability of Yersinia spp. to invade tissue culture cells is reflective of their ability to penetrate the intestinal epithelium and that this capacity is an important aspect of the disease process. Three different genes from Yersinia spp. that are involved in the tissue culture invasion phenotype have been identified: inv, ail, and yadA. It was previously shown that inv is necessary for efficient penetration of the intestinal epithelium by Yersinia enterocolitica. The present study was initiated to determine whether other known Yersinia invasion factors could promote uptake of the bacteria by mice in the absence of invasion. In addition, the roles of these three invasion factors in the survival of the bacteria, lethality for mice, and development of pathology were compared. We found that YadA is necessary for persistence of Y. enterocolitica in Peyer's patches, and consistent with this observation, the yadA mutant was avirulent for mice infected either orally or intraperitoneally. In addition, the inv yadA double mutant was avirulent. Histological and immunohistological examination of the Peyer's patches of infected mice indicated that despite the presence of large numbers of CFU at 24 h the yadA and ail yadA mutants cause only minimal pathology and recruitment of macrophages. At 42 h postinfection, Peyer's patches from mice infected with the inv mutant showed no pathology, despite the prediction that some of the mice by this time would be colonized. However, at 72 h, inflammation and necrosis were evident in some Peyer's patches. Together, these observations suggest that for visible pathology to develop, a threshold number of bacteria (> 10(5)) is needed and the bacteria need to persist for more than 24 h. Lastly, YadA but not Ail may play a role in the less efficient, delayed invasion of the intestinal epithelium observed for the inv mutant.

Animals↗
PubMed1995

In vitro and in vivo characterization of an ail mutant of Yersinia enterocolitica.

Ail is a 17-kDa protein of Yersinia enterocolitica previously identified on the basis of its ability to confer upon Escherichia coli the phenotype of attachment and invasion of cultured epithelial cells. Here we report an examination of the contribution of ail to the pathogenicity of Y. enterocolitica. A low-copy-number ail plasmid that promoted serum resistance in E. coli HB101 was constructed. The serum resistance phenotype conferred by ail to E. coli was affected by the growth phase of the culture as well as by the gene copy number. In contrast, the copy number of ail (and the relative quantity of Ail) was found to have little effect on the amount of Ail-promoted invasion of cultured epithelial cells. An ail mutant of Y. enterocolitica was constructed and characterized in vitro. This mutant produced no detectable Ail and had a reduced ability to invade CHO cells. Serum resistance of Y. enterocolitica was Ail dependent and was affected by growth phase and ail copy number. The phenotype of the ail mutant was examined in vivo by using a murine model for infection. The ail mutant phenotype was identical to that of the wild-type strain in oral 50% lethal dose studies and early colonization of Peyer's patches as well as in kinetic studies. Western blot (immunoblot) analysis of Ail produced by bacteria growing in vivo at 48 h postinfection indicated that ail was expressed at this time point. Thus, our findings confirm that Ail contributes to the serum resistance and invasion phenotypes of Y. enterocolitica in vitro and indicate that Ail is not required to establish an infection or to cause systemic infection of BALB/c or DBA/2 mice.

Animals↗
PubMed1995

Role of RpoS in survival of Yersinia enterocolitica to a variety of environmental stresses.

rpoS, a gene that encodes an alternative sigma factor (also known as katF), is critical for the ability of Yersinia enterocolitica grown at 37 degrees C, but not at 26 degrees C, to survive diverse environmental insults such as high temperature, hydrogen peroxide, osmolarity, and low pH. However, a Y. enterocolitica rpoS mutant was not affected in expression of inv or ail, invasion of tissue culture cells, or virulence in mice.

Adhesins, Bacterial↗
PubMed1994

Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene.

The inv gene encodes the protein invasin, which is the primary invasion factor for Yersinia enterocolitica in vitro and in vivo. Previous studies of Yersinia species have shown that inv expression and entry into mammalian cells are temperature regulated. Invasin production is reduced at the host temperature of 37 degrees C as compared to production at ambient temperature; consequently, this study was initiated to determine whether other host environmental signals might induce inv expression at 37 degrees C. An inv::phoA translational fusion was recombined on to the Y. enterocolitica chromosome by allelic exchange to monitor inv expression. Molecular characterization of expression of the wild-type inv gene and the inv::phoA fusion showed that invasin is not produced until early stationary phase in bacteria grown at 23 degrees C. Y. enterocolitica grown at 37 degrees C and pH 5.5 showed levels of inv expression comparable to those observed in bacteria grown at 23 degrees C. An increase in Na+ ions caused a slight increase in expression at 37 degrees C. However, expression at 37 degrees C was unaffected by anaerobiosis, growth medium, calcium levels, or iron levels. Additionally, Y. enterocolitica expressed invasin in Peyer's patches two days after being introduced intragastrically into BALB/c mice. These results suggest that invasin expression in Y. enterocolitica may remain elevated early during interaction with the intestinal epithelium, a site at which invasin was shown to be necessary.

Adhesins, Bacterial↗
PubMed1994

Mouse T lymphocytes that express a gamma delta T-cell antigen receptor contribute to resistance to Salmonella infection in vivo.

Mice depleted of lymphocytes expressing the alpha beta or the gamma delta T-cell receptor for antigen (TCR) by antibody treatment were infected orally with Salmonella enteritidis. In both groups of treated mice, the 50% lethal dose decreased, suggesting that both the alpha beta TCR+ and the gamma delta TCR+ subsets contribute to resistance to oral infection. These data provide further evidence for the contribution of gamma delta T cells in the response to bacterial infections.

Animals↗
PubMed1993

Cloning of the YenI restriction endonuclease and methyltransferase from Yersinia enterocolitica serotype O8 and construction of a transformable R-M+ mutant.

Two different clonal groups of pathogenic Yersinia enterocolitica strains, American and non-American, have been recognized. These are distinguished by a number of criteria, including their virulence in a murine model of infection. However, genetic analysis of virulence in American strains has been hampered due to the severe restriction of transformed or electroporated DNA. Thus, we cloned the yenIMR locus from the American serotype strain 8081c, which encodes YenI, an isoschizomer of PstI. This clone encodes both the restriction endonuclease and methyltransferase. The location of the genes on the clone was determined and this information was used to construct a small deletion (400 bp) that results in an R-M+ phenotype. This mutation was recombined onto the Y. enterocolitica chromosome to give an R-M+ mutant which showed at least a 1000-fold increase in electroporation frequency compared to the wild-type strain. Southern analysis using a probe derived from yenIMR indicated that American serotype strains have this locus whereas non-American serotype strains do not.

Chromosome Mapping↗
PubMed1993

Yersinia enterocolitica invasin: a primary role in the initiation of infection.

The ability to invade the intestinal epithelium of mammals is an essential virulence determinant of Yersinia enterocolitica. The chromosomally encoded Y. enterocolitica 8081v invasion gene, inv, was disrupted to assess its role in pathogenesis. The inv mutant (JP273v) was approximately 80-fold less invasive than wild type for cultured epithelial cells. When mice were infected intragastrically, up to 10(7) fewer JP273v were recovered from Peyer's patches early (6-18 hr) after infection compared with wild type. Analysis of the course of infection revealed that the inv mutant had distinct differences relative to wild type in the distribution of visible infectious foci and in tissue colonization; however, the mutant and wild-type strains had similar LD50 values for both orally and intraperitoneally infected mice. The invasion defect of the inv mutant was fully complemented in vitro and in vivo by introduction of the wild-type inv gene in trans. The inv gene product, invasin, appears to play a vital role in promoting entry during the initial stage of infection. During the subsequent establishment of a systemic infection, invasin may be of secondary importance, since the Y. enterocolitica inv mutant was as proficient as wild type at causing a fatal infection in mice. Based on these data, we discuss the role of invasin in a naturally occurring Y. enterocolitica infection.

Adhesins, Bacterial↗
PubMed1993

Stress induction of the virulence proteins (SpvA, -B, and -C) from native plasmid pSDL2 of Salmonella dublin.

The virulence region of the wild-type plasmid pSDL2 contained in Salmonella dublin is highly conserved among plasmids from several nontyphoid Salmonella serotypes and is essential for the development of systemic infection in BALB/c mice. Polyclonal antibodies against three proteins (SpvA, -B, and -C) expressed from a 4.1-kb EcoRI subclone of the plasmid virulence region were generated. These antibodies were used to detect expression of the Spv proteins when S. dublin was grown in vitro under stress-inducing conditions, such as nutrient deprivation and increased temperature, that the bacteria may encounter during the course of infection within the host. Glucose starvation resulted in expression of all three proteins shortly after the lag phase. When the bacteria were grown to the late-log phase without glucose, heat shock strongly induced expression of SpvA but not SpvB or SpvC. The addition of 0.2% glucose to the medium resulted in loss of expression of the proteins until the late-log to stationary phase. Iron limitation or lowered pH induced expression of the proteins during exponential growth even in the presence of glucose. Insertion mutations into the positive regulator gene spvR upstream from spvABC and insertions into spvA and spvC resulted in loss of expression of SpvA, -B, and -C, suggesting a complex regulation of expression. These studies define a variety of environmental conditions that induce expression of the Spv virulence proteins from the wild-type plasmid pSDL2 in S. dublin in vitro.

Animals↗
PubMed1993

The biological role of invasin during a Yersinia enterocolitica infection.

Epidemiological data suggest that the ability to invade the intestinal epithelium of mammals is an essential virulence determinant of Yersinia enterocolitica. The chromosomally encoded Y. enterocolitica 8081v invasion gene, inv, was disrupted to assess its role in pathogenesis. The inv mutant was unable to invade cultured epithelial cells as efficiently as wild type. Furthermore, when mice were infected intragastrically, the inv mutant was extremely deficient at penetrating the murine intestinal epithelium. Analysis of the course of infection showed that the inv mutant had distinct differences relative to wild type in the distribution of visible infectious foci and in tissue colonization; however, the mutant and wild-type strains had similar median lethal doses for both orally and intraperitoneally infected mice. The invasion defect of the inv mutant was fully complemented in vitro and in vivo by introduction of the wild-type inv gene in trans. The inv gene product, invasin, appears to play a vital role in promoting entry during the initial stage of infection. During the subsequent establishment of a systemic infection, invasin may be of secondary importance, since the Y. enterocolitica inv mutant was as proficient as wild-type at causing a fatal infection in mice. The possible role of invasin in a naturally occurring Y. enterocolitica infection is discussed.

Adhesins, Bacterial↗
PubMed1992

Clinical and microbiologic characteristics of cutaneous infection with Yersinia enterocolitica.

The clinical and microbiologic features of primary cutaneous infections by Yersinia enterocolitica are described in three children. Vesiculobullous lesions developed in two patients, and an intense granulation response followed incision and drainage. In the third child, cellulitis and abscess formation developed at the site of minor skin trauma. Y. enterocolitica isolates from the patients were extensively serologically, biochemically, and molecularly analyzed and compared with virulent Y. enterocolitica strains. The ability of the isolates to adhere to and invade eukaryotic cells was determined using in vitro assays; virulence was assessed by inoculation of suckling mice. The resulting data suggest that primary cutaneous infections by Y. enterocolitica involve strains that are as virulent as pathogenic gastrointestinal isolates.

Animals↗
PubMed1992

An unusual pagC::TnphoA mutation leads to an invasion- and virulence-defective phenotype in Salmonellae.

Two phenotypes believed to contribute to the pathogenesis of Salmonella infections are macrophage survival and invasion of epithelial cells. It was recently observed that the Salmonella macrophage survival factor PagC has significant amino acid similarity to the Yersinia invasion factor Ail. This observation raised the possibilities that macrophage survival is in part determined by the pathway of entry and that PagC confers an entry mechanism that does not trigger the microbicidal activities of the macrophage. Thus, we sought to investigate the role of PagC in invasion by examining (i) the invasion phenotype of pagC mutants and (ii) the invasion phenotype of Escherichia coli carrying pagC. A previously identified invasion-defective TnphoA insertion mutant of Salmonella enteritidis was found to have TnphoA inserted into the signal sequence-encoding region of pagC; the pagC allele from this mutant, SM5T, was designated pagC64. In contrast, Salmonella typhimurium carrying the pagC1 allele (a TnphoA insertion mutation, downstream of the region encoding the signal sequence) was not defective for invasion. Further analysis of these two pagC alleles suggested that the invasion-defective phenotype associated with pagC64 is not due to the loss of PagC function but rather is due to the synthesis of a hybrid PagC-alkaline phosphatase protein that is aberrantly localized, most likely to the inner membrane, and thus may prevent proper localization or function of a factor(s) required for efficient invasion. The observation that pagC did not confer an invasive phenotype to E. coli further suggests that PagC is not an invasion factor. A cloned pagC gene complemented the macrophage survival defect of S. typhimurium pagC1 mutants, but the cloned ail gene did not. Together these results suggest that the structural similarity between PagC and Ail may not extend to a similarity in function. Interestingly, S. enteritidis carrying the pagC64 allele that results in both an invasion defect and a macrophage survival defect was less virulent for mice infected intragastrically or intraperitoneally than was S. enteritidis carrying the pagC1 allele that results only in a macrophage survival defect.

Alkaline Phosphatase↗
PubMed1992

Identification of novel loci affecting entry of Salmonella enteritidis into eukaryotic cells.

There are an estimated 2 million cases of salmonellosis in the United States every year. Unlike the incidence of many infectious diseases, the incidence of salmonellosis in the United States and other developed countries has been rising steadily over the past 30 years, and the disease now accounts for 10 to 15% of all cases of acute gastroenteritis in the United States. The infecting organism is ingested and must traverse the intestinal epithelium to reach its preferred site for multiplication, the reticuloendothelial system. Despite several recent studies, the genetic basis of the invasion process is poorly understood. An emerging theme from these studies is that wild-type Salmonella organisms probably have several chromosomal loci that are required for the most efficient level of invasion. In this study, we have identified and characterized 13 TnphoA insertion mutants of Salmonella enteritidis CDC5 that exhibit altered invasion phenotypes. The mutants were identified by screening a bank of TnphoA insertions in S. enteritidis CDC5str for their invasion phenotype in three tissue culture cell lines (HEp-2, CHO, and MDCK). These 13 mutants were separated into six classes based on their invasive phenotypes in the tissue culture cell lines. Several mutants were defective for entry of some cell lines but not for others, while two mutants (SM6 and SM7) were defective for entry into all three tissue culture cell lines. This suggests that Salmonella spp. may express more than one invasion pathway. Southern analysis and chromosomal mapping indicated that as many as nine chromosomal loci may contribute to the invasion phenotype. It is becoming clear that the invasive phenotype of Salmonella spp. is multifactorial and more complex than that of some other invasive members of the family Enterobacteriaceae.

Animals↗