Jeffrey D. Hillman

Characterization and Pathogenic Significance of Vibrio vulnificus Antigens Preferentially Expressed in Septicemic Patients

ABSTRACT Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescent-phase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methyl-accepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and IlvC), secretion (TatB and plasmid Achromobacter secretion [PAS] factor), transcriptional activation (IlvY and HlyU), and the activity of a putative lipoprotein (YaeC). In addition, one identified open reading frame encoded a hypothetical protein. Isogenic mutants of the 12 in vivo-expressed (ive) genes were constructed and tested for cytotoxicity. Cytotoxic activity of the mutant strains, as measured by lactate dehydrogenase release from HeLa cells, was nearly abolished in pyrH, purH, and hlyU mutants. The intraperitoneal 50% lethal dose in mice increased by ca. 10- to 50-fold in these three mutants. PyrH and PurH seem to be essential for in vivo growth. HlyU appears to be one of the master regulators of in vivo virulence expression. The successful identification of ive genes responsible for the in vivo bacterial virulence, as done in the present study, demonstrates the usefulness of IVIAT for the detection of new virulence genes.

Use of in vivo-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae

In vivo-induced antigen technology is a method to identify proteins expressed by pathogenic bacteria during human infection. Sera from 10 patients convalescing from cholera infection in Bangladesh were pooled, adsorbed against in vitro-grown El Tor Vibrio cholerae O1, and used to probe a genomic expression library in Escherichia coli constructed from El Tor V. cholerae O1 strain N16961. We identified 38 positive clones in the screen, encoding pili (PilA and TcpA), cell membrane proteins (PilQ, MshO, MshP, and CapK), methyl-accepting chemotaxis proteins, chemotaxis and motility proteins (CheA and CheR), a quorum-sensing protein (LuxP), and four hypothetical proteins. Analysis of immune responses to purified PilA and TcpA in individual patients demonstrated that the majority seroconverted to these proteins, confirming results with pooled sera. These results suggest that PilA and its outer membrane secretin, PilQ, are expressed during human infection and may be involved in colonization of the gastrointestinal tract. These results also demonstrate substantial immune responses to TcpA in patients infected with El Tor V. cholerae O1. In vivo-induced antigen technology provides a simple method for identifying microbial proteins expressed during human infection, but not during in vitro growth.

Genetically modified Streptococcus mutans for the prevention of dental caries

There are many examples of positive and negative interactions between different species of bacteria inhabiting the same ecosystem. This observation provides the basis for a novel approach to preventing microbial diseases called replacement therapy. In this approach, a harmless effector strain is permanently implanted in the host’s microflora. Once established, the presence of the effector strain prevents the colonization or outgrowth of a particular pathogen. In the case of dental caries, replacement therapy has involved construction of an effector strain called BCS3-L1, which was derived from a clinical Streptococcus mutans isolate. Recombinant DNA technology was used to delete the gene encoding lactate dehydrogenase in BCS3-L1 making it entirely deficient in lactic acid production. This effector strain was also designed to produce elevated amounts of a novel peptide antibiotic called mutacin 1140 that gives it a strong selective advantage over most other strains of S. mutans. In laboratory and rodent model studies, BCS3-L1 was found to be genetically stable and to produce no apparent deleterious side effects during prolonged colonization. BCS3-L1 was significantly less cariogenic than wild-type S. mutans in gnotobiotic rats, and it did not contribute at all to the cariogenic potential of the indigenous flora of conventional Sprague-Dawley rats. And, its strong colonization properties indicated that a single application of the BCS3-L1 effector strain to human subjects should result in its permanent implantation and displacement over time of indigenous, disease-causing S. mutans strains. Thus, BCS3-L1 replacement therapy for the prevention of dental caries is an example of biofilm engineering that offers the potential for a highly efficient, cost effective augmentation of conventional prevention strategies. It is hoped that the eventual success of replacement therapy for the prevention of dental caries will stimulate the use of this approach in the prevention of other bacterial diseases.

IVIAT: a novel method to identify microbial genes expressed specifically during human infections

In vivo induced antigen technology (IVIAT) is a novel technology that can quickly and easily identify in vivo induced genes in human infections, without the use of animal models. This technology is expected to facilitate the discovery of new targets for vaccines, antimicrobials and diagnostic strategies in a wide range of microbial pathogens.

Construction and characterization of an effector strain of streptococcus mutans for replacement therapy of dental caries.

ABSTRACT An effector strain has been constructed for use in the replacement therapy of dental caries. Recombinant DNA methods were used to make theStreptococcus mutans supercolonizing strain, JH1140, lactate dehydrogenase deficient by deleting virtually all of theldh open reading frame (ORF). To compensate for the resulting metabolic imbalance, a supplemental alcohol dehydrogenase activity was introduced by substituting the adhB ORF fromZymomonas mobilis in place of the deleted ldhORF. The resulting clone, BCS3-L1, was found to produce no detectable lactic acid during growth on a variety of carbon sources, and it produced significantly less total acid due to its increased production of ethanol and acetoin. BCS3-L1 was significantly less cariogenic than JH1140 in both gnotobiotic- and conventional-rodent models. It colonized the teeth of conventional rats as well as JH1140 in both aggressive-displacement and preemptive-colonization models. No gross or microscopic abnormalities of major organs were associated with oral colonization of rats with BCS3-L1 for 6 months. Acid-producing revertants of BCS3-L1 were not observed in samples taken from infected animals (reversion frequency, <10−3) or by screening cultures grown in vitro, where no revertants were observed among 105 colonies examined on pH indicator medium. The reduced pathogenic potential of BCS3-L1, its strong colonization potential, and its genetic stability suggest that this strain is well suited to serve as an effector strain in the replacement therapy of dental caries in humans.

Colonization of the Human Oral Cavity by a Streptococcus mutans Mutant Producing Increased Bacteriocin

Streptococcus mutans strain JH1005 is a mutant that produces levels of bacteriocin activity three-fold-elevated than those produced by its parent, JH1001. A single infection regimen with JH1005 was found to result in persistent colonization of the teeth of all three adult subjects tested. This is a significant improvement over JH1001, which required multiple exposures in order to colonize the teeth of humans reliably. The levels of total cultivable bacteria and indigenous S. sanguis were not affected by JHI005 colonization. In two of the three subjects, total (indigenous plus JH1005) S. mutans levels were significantly decreased. The results provide additional support for the role of bacteriocin production as an ecological determinant in colonization by S. mutans. They also indicate that a practical regimen for infection by an effector strain might be achieved for use in the replacement therapy of dental caries.

Microarray analysis of quorum-sensing-regulated genes in Porphyromonas gingivalis

ABSTRACT Quorum sensing is a phenomenon defined as gene regulation in response to cell density that regulates various functions in bacteria. The periodontopathogen Porphyromonas gingivalis possesses a luxS gene homologue that may encode a quorum-sensing system. In order to identify genes of P. gingivalis that are regulated by luxS, gene expression analysis was done using microarrays and RNA samples from the W83 wild-type strain and an isogenic luxS mutant, LY2001. The results indicated that 17 open reading frames (ORFs) in LY2001 are upregulated and two are downregulated. Real-time PCR was done to confirm the microarray results. Among the upregulated ORFs is a group of stress-related genes, including htrA, clpB, groEL, dnaK, and the F subunit of alkyl hydroperoxide reductase. This suggested that luxS is involved in stress gene regulation in P. gingivalis. Stress response experiments, including high-temperature survival, resistance to hydrogen peroxide (H2O2), and survival during exposure to low and high pH, were performed on the P. gingivalis wild-type and LY2001 strains. LY2001 had a significantly higher survival rate than did W83 when stressed at 50°C. No difference was found at pH 5, but LY2001 had increased survival compared to W83 at pH 9. LY2001 also survived better than W83 when stressed with 0.35 mM H2O2. These results suggest that luxS might be involved in promoting survival of P. gingivalis in the host by regulating its response to host-induced stresses such as temperature, H2O2, and pH.

In vivo induced antigen technology (IVIAT)

In vivo induced antigen technology (IVIAT) is a technique that identifies pathogen antigens that are immunogenic and expressed in vivo during human infection. IVIAT is complementary to other techniques that identify genes and their products expressed in vivo. Genes and gene pathways identified by IVIAT may play a role in virulence or pathogenesis during human infection, and may be appropriate for inclusion in therapeutic, vaccine or diagnostic applications.

Identification of Candida albicans genes induced during thrush offers insight into pathogenesis

Candida albicans causes a wide spectrum of diseases, ranging from mucocutaneous infections like oral thrush to disseminated candidiasis. Screening for C. albicans genes expressed within infected hosts might advance understanding of candidal pathogenesis, but is impractical using existing techniques. In this study, we used an antibody‐based strategy to identify C. albicans genes expressed during thrush. We adsorbed sera from HIV‐infected patients with thrush against candidal cells grown in vitro and screened a C. albicans genomic expression library. We identified 10 genes encoding immunogenic antigens and used reverse transcription‐polymerase chain reaction to verify that they were induced within thrush pseudomembranes recovered from a patient. The in vivo induced genes are involved in diverse functions, including regulation of yeast‐hyphal morphogenesis, adhesion to host cells, nutrient uptake, phospholipid biosynthesis and amino acid catabolism. Four genes encode known virulence determinants (HWP1, CST20, CPP1 and RBF1). Another gene, LPD1, for which a role in candidal pathogenesis is unknown, encodes a protein homologous to a bacterial virulence determinant. Most importantly, disruption of CaNOT5, a newly identified gene, conferred defects in morphogenesis, decreased adherence to human buccal epithelial cells and attenuated mortality during murine disseminated candidiasis, proving that our strategy can identify genes encoding novel virulence determinants.

Use of in vivo-induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection

ABSTRACT Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.