Dwayne C. Savage

Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species

Lactobacillus johnsonii strain 100-100 expresses two antigenically distinct conjugated bile salt hydrolases (BSH), alpha and beta, that combine to form native homo- and heterotrimers. This paper reports characterization of loci within the genome that encode this capacity. A locus that encodes BSH beta (cbsH beta), a partial (cbsT1) and a complete conjugated bile salt transporter (cbsT2) was identified previously. DNA sequence analysis at this locus was extended and revealed a complete ORF for cbsT1 and no other ORFs in tandem. The three genes, cbsT1, cbsT2 and cbsH beta, probably constitute an operon; a putative promoter was identified upstream of cbsT1. A second locus that expresses BSH activity in strain 100-100 was identified. Sequence analysis of the clone predicted a 978 nt ORF that did not share tandem organization with other ORFs, was similar in sequence to other BSH genes, and matched, in predicted protein sequence, the first 25 amino acids of BSH alpha. A phenotypic screen for BSH activity and a genetic screen for the cbsH beta locus were performed on 50 Lactobacillus isolates from humans or dairy products. Nearly all of the isolates that were positive for cbsH beta were from human sources. Variability in the BSH phenotype and cbsH beta genotype was identified in isolates of the same species. DNA sequence was obtained and analysed from the cbsH beta locus of one human isolate, L. acidophilus strain KS-13. This organism has cbsT1, cbsT2 and cbs beta genes that are 84, 87 and 85% identical in DNA sequence to those of strain 100-100. DNA sequence identity to strain 100-100 ends in regions flanking this locus. The findings of this study suggest that BSH genes have been acquired horizontally and that BSH activity is important at some level for lactobacilli to colonize the lower gastrointestinal tract.

Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33

Lactobacillus sp. strain 100-33 is resistant to macrolides, lincosamides, and streptogramin B-type antibiotics (MLSR) and appears to contain several major and minor plasmids. One of these plasmids, pLAR33, is approximately 18 kbp in size. When cells of strain 100-33 were protoplasted and regenerated, an MLSS isolate was derived. The derivative, designated strain ES1, contained a unique plasmid complement in which it had apparently lost the major plasmids of the parental strain, including pLAR33, and retained only a minor plasmid seen in low concentrations in strain 100-33. The MLSR determinant was cloned from plasmid DNA of strain 100-33 on a 3-kbp EcoRV fragment into pBR322 and localized to pLAR33. The determinant expressed macrolide and lincosamide resistance in Escherichia coli HB101, was localized to approximately 1 kbp on the cloned sequence, and is apparently under the control of its own promoter. MLSR electroporants were derived from strain ES1 electroporated with plasmid DNA from strain 100-33; these MLSR isolates had acquired a plasmid complement similar to that of strain 100-33, including pLAR33. Endonuclease digestion and Southern analysis of plasmid DNA from both strains indicated that the major plasmids are multimeric and deleted forms of one archetypal extrachromosomal element.

Adhesion of Candida albicans to epithelial cells effect of polyoxin D

Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.

CbsT2 from Lactobacillus johnsonii 100-100 Is a Transport Protein of the Major Facilitator Superfamily That Facilitates Bile Acid Antiport

We previously identified two conjugated bile acid transporters, CbsT1 and CbsT2, in Lactobacillus johnsonii 100-100 and Lactobacillus acidophilus KS-13 that are gene duplicates encoded in tandem with a conjugated bile salt hydrolase (BSH) [Elkins and Savage, J. Bacteriol. 180:4344–4349, 1998; Elkins et al., Microbiology 147: 3403–3412, 2001]. CbsT2 from 100–100 was shown to increase taurocholic acid (TCA) uptake in Escherichia coli; however, higher levels were achieved when an extracellular factor (EF) from 100–100 was present in the assay medium (spent medium from 100–100, pH 4.2). We continued this study here to determine the role of EF in this transport system. Kinetic studies revealed that the previously observed CbsT2- and EF-mediated TCA accumulation is rapid (<15 s) but not saturable, suggesting that EF is limiting. In addition, uptake of TCA by E. coli expressing CbsT2 was insensitive to ionophores, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, and thus, is independent of the proton motive force. Since BSH converts [24-14C]TCA to [24-14C]cholic acid (CA), we measured net radiolabel uptake in E. coli cells expressing transporter(s) and BSH. Interestingly, such cells accumulated less 14C radiolabel (by approximately half) than cells expressing CbsT2 alone. These data can be explained if CA diffuses out of E. coli through the transporter(s). We, therefore, added exogenous, unlabeled CA to EF-spent media, which under our assay conditions, performed similarly to EF+ culture supernatant in TCA and CA uptake assays. Thus, unlabeled CA (a protonated, neutral lipophile) can partition directly into E. coli cells especially at low pH. These findings were validated in uptake assays with [3H]TCA, which yields [3H]taurine (a hydrophilic moiety) upon hydrolysis by the BSH. Amounts of cell-associated 3H radiolabel remained similar in cells expressing CbsT2 and BSH versus cells expressing only CbsT2, both of which were higher than in cells expressing BSH alone. Our data support a hypothesis that these transporters, which comprise a new subfamily of the major facilitator superfamily, facilitate antiport of TCA and CA.

Chapter 2 – Mucosal Microbiota

This chapter provides information on the microbial populations on normal mucosal surfaces in humans (the indigenous microbiota). It also evaluates some evidence on how such populations interact with immunologic functions. Microorganisms, principally bacteria, colonize the skin and mucosal surfaces in most areas of the adult human body. Gram-positive, aerobic bacteria predominate on the skin and the mucosa of the nasopharynx and oropharynx. Gram-positive and gram-negative facultative and anaerobic bacteria also colonize the mucosal surface and lumen of major portions of the gastrointestinal (GI) tract. The anaerobic bacteria predominate in both the buccal cavity, the distal one-third of the small intestine, and large intestine. Functional anaerobes also predominate on vaginal surfaces. In aggregate, the microbial populations on all the mucosal surfaces exceed 100 trillion (1*10 14 ) cells. This population, over 99% of which is found in the large intestine, constitutes an indigenous microbiota. This biota establishes on the various mucosal surfaces after birth in patterns that can be recognized as ecologic successions. The indigenous microbiota functions in “colonization resistance” to inhibit the nonindigenous microorganisms and certain indigenous microbial pathogens from proliferating in the mucosal habitats.