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Most HIV transmission occurs on the mucosal surfaces of the gastrointestinal and cervicovaginal tracts, both of which are normally coated by a biofilm of nonpathogenic commensal bacteria. We propose to genetically engineer such naturally occurring bacteria to protect against HIV infection by secreting antiviral peptides. Here we describe the development and characterization of Nissle 1917, a highly colonizing probiotic strain of Escherichia coli , secreting HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells. By using an appropriate combination of cis- and transacting secretory and regulatory signals, micromolar secretion levels of the anti-HIV peptides were achieved. The genetically engineered Nissle 1917 were capable of colonizing mice for periods of weeks to months, predominantly in the colon and cecum, with lower concentrations of bacteria present in the rectum, vagina, and small intestine. Histological and immunocytochemical examination of the colon revealed bacterial growth and peptide secretion throughout the luminal mucosa and in association with epithelial surfaces. The use of genetically engineered live microbes as anti-HIV microbicides has important potential advantages in economy, efficacy, and durability.

A temperate bacteriophage isolated from Vibrio cholerae O139, the new epidemic strain of cholera, was found to have a polyhedral head 65 nm in diameter and a rigid contractile tail 120 nm in length. The phage chromosome was a double-stranded DNA of 35 kb, with unique cohesive ends and had a G + C content of 58.8%. A restriction map of the phage DNA was constructed using the restriction endonucleases AvaI and BstEII. The phage, whose presence could be detected in nine out of 13 V. cholerae O139 isolates tested, was found to have identical chromosomal integration sites in all the strains. The phage attachment site (attP) was found to be located very close to one end of the genome.

Four outbreak strains of Vibrio cholerae O139 from endemic areas of India and Bangladesh were found to carry lysogenic phage(s). All of these phage(s) produced turbid plaques characteristic of lysogeny on V. cholerae MAK 757 (El Tor, Ogawa) cells as well as on their VcA-1 lysogens but were unable to infect V. cholerae 154 (classical) cells, the universal host for all classical phages. Colonies in the turbid plaques were O139 lysogens and these developed an auxotrophic requirement, mainly for purines suggesting the integration of the prophage into the host chromosome. The immunity profile of the O139 phage(s) was similar to that of phage alpha but differed in the sensitivity of the phage lysogen of V. cholerae MAK 757 to subsequent infection by phage beta.

Summary. Four lines of evidence suggest that the recent outbreak strains of Vibrio cholerae 0139 could have emerged from serogroup 01 strains typified by isolates MOl and M0477 described in this paper, which are neither truly classical nor truly El Tor in their biotype attributes. Firstly, like all 0139 isolates, these 01 strains, isolated in Madras during and before the 0139 outbreak, were resistant not only to polymyxin B but also to all biotypespecific choleraphages, i.e. classical phage (Dl49 and El Tor phages e4 and e5. Secondly, the restriction fragment pattern (RFP) polymorphism displayed by these strains for the cholera toxin (ctx) gene, were identical with those produced by 0139 isolates but were different from those of 01 type strains, namely V. cholerae 569B (classical) and V. cholerae MAK757 (El Tor). Thirdly, all the 0139 isolates and the two 01 isolates carried an identical large number of copies of cholera toxin gene in their chromosomes. Finally, the outer-membrane protein profiles of strains MO 1 and M0477 were identical to those of 0 139 isolates but were different from those displayed by strains 569B and MAK757.

The status of E. coli K-12 as an exclusively non-invasive, non-pathogenic bacterium has almost been incontrovertible. Our recent finding that a mutation in one of its main architectural protein, HU, converts E. coli K-12 to an actively invasive form suggests that gaining host cell entry might be an expedient survival tactic for traditional commensals during certain altered host conditions. The mutant E. coli (SK3842) exhibits properties usually associated with pathogenic bacteria: host cell invasion, phagosomal disruption and intracellular replication. However, unlike the situation with some pathogens, internalized SK3842 imparts anti-apoptotic and cyto-protective effects rather than lethality on the host cell, both in vitro and in vivo. Here, we show that SK3842 also provides colonization resistance against other invasive pathogens--a trait not shared by the parental commensal strain. Thus, the altered lifestyle of SK3842 encompasses characteristics both from traditional pathogens as well as beneficial probiotic strains.

The HUαE38K, V42L mutant of the bacterial histone-like protein HU causes a major change in the transcription profile of the commensal organism Escherichia coli K-12 (Kar S, Edgar R, Adhya S, Proc. Natl. Acad. Sci. U. S. A. 102:16397–16402, 2005). Among the upregulated genes are several related to pathogenic interactions with mammalian cells, as evidenced by the expression of curli fibers, Ivy, and hemolysin E. When E. coli K-12/ HUαE38K, V42L was added to Int-407 cells, there was host cell invasion, phagosomal disruption, and intracellular replication. The invasive trait was also retained in a murine ileal loop model and intestinal explant assays. In addition to invasion, the internalized bacteria caused a novel subversion of host cell apoptosis through modification and regulation of the BH3-only proteins BimEL and Puma. Changes in the transcription profile were attributed to positive supercoiling of DNA leading to the altered availability of relevant promoters. Using the E. coli K-12/HUαE38K, V42L variant as a model, we propose that traditional commensal E. coli can adopt an invasive lifestyle through reprogramming its cellular transcription, without gross genetic changes., IMPORTANCE Escherichia coli K-12 is well established as a benign laboratory strain and a human intestinal commensal. Recent evidences, however, indicate that the typical noninvasive nature of resident E. coli can be reversed under specific circumstances even in the absence of any major genomic flux. We previously engineered an E. coli strain with a mutant histone-like protein, HU, which exhibited significant changes in nucleoid organization and global transcription. Here we showed that the changes induced by the mutant HU have critical functional consequences: from a strict extracellular existence, the mutant E. coli adopts an almost obligate intracellular lifestyle. The internalized E. coli exhibits many of the prototypical characteristics of traditional intracellular bacteria, like phagosomal escape, intracellular replication, and subversion of host cell apoptosis. We suggest that E. coli K-12 can switch between widely divergent lifestyles in relation to mammalian host cells by reprogramming its cellular transcription program and without gross changes in its genomic content.

We studied the restriction fragment length polymorphism of the rRNA gene and CTX genetic element in Vibrio cholerae O139 Bengal, which resurged in Calcutta in September 1996 after a gap of 32 months. While the strains from this resurgence were indistinguishable from the earlier strains by ribotyping, the structure of the CTX genetic element present in the current O139 strains was found to be unconventional.

An unusual filamentous bacteriophage, VSK, containing single-stranded, circular DNA as its genome was isolated from Vibrio cholerae 0139 strains P07 and B04. Unlike other single-stranded DNA phages, VSK can integrate its genome into the chromosome of the host and enter into a lysogenic state. The double-stranded replicative form (RF) of the single-stranded phage DNA was isolated. A restriction map of the VSK RF DNA was constructed using HaeII, AvaII, ClaI and XbaI. By Southern blot analysis of the chromosomal DNA of the lysogen using labeled phage DNA as probe, the attachment site (attP) on the viral genome was also identified.

SummarySixty-nine strains ofVibrio cholerae01 isolated at different times were analysed to investigate if there were any differences among the 01 strains isolated before, during and after the advent of the O139 serogroup. Of the 69 01 strains examined, 68 belonged to the Ogawa serotype while one belonged to the Inaba serotype. With the exception of one strain all other strains ofV. cholerae01 belonged to the eltor biotype. A single O1 strain isolated before the emergence of the O139 serogroup could not be classified as either eltor or classical biotype because it was resistant to both classical and eltor specific bacteriophages. Marked variations in the susceptibility to antibiotics ofV. choleraeO1 isolated during the different periods were observed. In addition, strains ofV. choleraeisolated after the epidemic of serogroup O139 in Calcutta showed an expanding R-type with resistance to a variety of drugs as compared to the 01 strains isolated before the advent of the O139 serogroup. From this study, it is clear that there is a substantial mobility in genetic elements ofV. choleraeOl which necessitates a continuous monitoring to keep abreast of the changing traits of the etiologic agent of cholera.