Construction and characterization of an auxotrophic ctxA mutant of O139 Vibrio cholerae.

Cholera caused by the O139 serogroup still remains a public health concern in certain regions of the world and the existing O1 vaccines do not cross-protect cholera caused by this serogroup. An aminolevulinic acid (ALA) auxotroph vaccine candidate against the O139 serogroup, designated as VCUSM2, was recently developed. It was found to be immunogenic in animal model studies but showed mild reactogenic effects due to the presence of two intact copies of Vibrio cholerae toxin (CTX) genetic element. In the present study we have modified the ctx operon by systematic allelic replacement methodology to produce a mutant strain, designated as VCUSM14. This strain has two copies of chromosomally integrated and mutated ctxA gene, encoding immunogenic but not toxic cholera toxin A subunit (CT-A). The amino acids arginine and glutamic acid at position 7th and 112th, respectively, in CT-A of VCUSM14 were substituted with lysine (R7K) and glutamine (E112Q), respectively. Two copies of the ace and zot genes present in the ctx operon were also deleted. Cholera toxin-ELISA using GM1 ganglioside showed that the both wild type CT and mutated CT were recognized by anti-CT polyclonal antibodies. VCUSM14 produced comparatively less amount of antigenic cholera toxin when compared to the VCUSM2 and Bengal wild type strain. VCUSM14 did not elicit fluid accumulation when inoculated into rabbit ileal loops at doses of 10(6) and 10(8) CFU. The colonization efficiency of VCUSM14 was one log lower than the parent strain, VCUSM2, which can be attributed to the ALA auxotrophy and less invasive properties of VCUSM14. VCUSM14, thus a non-reactogenic auxotrophic vaccine candidate against infection by O139 V. cholerae.

Molecular epidemiology of tuberculosis in Malaysia.

Molecular typing with IS6110 was applied to Mycobacterium tuberculosis isolates from all parts of Malaysia. The degree of clustering increased with patient age, suggesting that reactivation may contribute to clustering. Identical banding patterns were also obtained for isolates from widely separate regions. Therefore, the use of clustering as a measure of recent transmission must be treated with caution. Strains related to the Beijing family were common in Peninsular Malaysia but were less common in Sabah and Sarawak, while a distinct group of strains comprised nearly 40% of isolates from East Malaysia but such strains were rare in Peninsular Malaysia. Single-copy strains, common in South and Southeastern Asia, constituted nearly 20% of isolates from the peninsula but were virtually absent in East Malaysia. The marked geographical difference in the prevailing strains indicates not only a restricted dissemination of M. tuberculosis but also a considerable degree of stability in the banding patterns.

Conservation of IS6110 sequence in strains of Mycobacterium tuberculosis with single and multiple copies.

The insertion sequence IS6110/IS986 is used extensively for detecting and typing Mycobacterium tuberculosis. In order to appreciate the evolutionary and epidemiological significance of differences in the fingerprint pattern, it is necessary to understand the factors influencing transposition frequency. Although most strains have many copies of this element, some have only one or two copies; it has been suggested that this apparent transpositional defect arises from the minor differences in the sequence of the element from different strains. In contrast, we have found that the sequence is identical in high and low copy number strains, indicating that external factors, such as the presence of adjacent promoters, must be responsible for differences in copy number.

Insertion sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110.

DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.

Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family.

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).

Does Mycobacterium tuberculosis have plasmids?

Evidence of the presence of plasmids in Mycobacterium tuberculosis is lacking, whereas they are widespread in some other mycobacterial species. We examined, by agarose gel electrophoresis, a total of 197 clinical isolates of M. tuberculosis, mostly resistant to one or more antibiotics, and were able to detect bands of apparently extrachromosomal DNA at a low level in some isolates. These presumptive plasmids could not be isolated by CsCl/ethidium bromide gradient ultracentrifugation, and may consist of unusual forms of DNA. The possible existence of single stranded plasmid DNA is discussed.

Polymorphic repetitive DNA sequences in Mycobacterium tuberculosis detected with a gene probe from a Mycobacterium fortuitum plasmid.

Clinical isolates of Mycobacterium tuberculosis were shown by Southern blotting to contain DNA sequences hybridizing to a probe derived from a Mycobacterium fortuitum plasmid. Two such M. tuberculosis DNA fragments, isolated from a gene library, were used as probes to show restriction fragment length polymorphism in M. tuberculosis strains by detecting a repetitive sequence apparently located at different points on the chromosome. This could indicate the presence of a transposable element in M. tuberculosis which is partly homologous to a region of the M. fortuitum plasmid. The probes described can be used to fingerprint M. tuberculosis isolates, and in addition are capable of distinguishing M. tuberculosis from Mycobacterium bovis and BCG.

Transformation of Mycobacterium smegmatis with E. coli plasmids.

One limiting factor in the studies of tuberculosis and leprosy is the lack of a versatile system for genetic analysis and manipulation of mycobacteria. One strategy used in constructing a plasmid vector for transforming Mycobacterium smegmatis was to insert fragments of a mycobacterial plasmid into an Escherichia coli plasmid. We found that the parental E. coli plasmid is capable of self-replication in M. smegmatis yielding chloramphenicol-resistant colonies. Plasmids from different passages of one M. smegmatis transformant were recovered and characterised by restriction digest analysis. The plasmid from the earlier passage was found to be indistinguishable from the original plasmid by restriction analysis. Plasmids from later preparations, however, were found to have undergone modifications in the M. smegmatis host resulting in an apparent increase in transformation efficiency for M. smegmatis. These plasmids can be used as a shuttle vector for the genetic manipulation of mycobacterial species.

Transformation of Mycobacterium smegmatis with Escherichia coli plasmids carrying a selectable resistance marker.

One limiting factor in studies of tuberculosis and leprosy is the difficulty of genetic analysis and manipulation of mycobacteria. Two approaches were adopted for the construction of vectors, based on different Escherichia coli plasmids and using Mycobacterium smegmatis as the host. In both cases we found that the original E. coli plasmid is capable of being replicated in M. smegmatis, yielding chloramphenicol-resistant colonies. One such plasmid has been recovered from a M. smegmatis transformant and used to re-transform both M. smegmatis and E. coli to chloramphenicol resistance. This plasmid is indistinguishable from the original plasmid by restriction analysis, and can be used as a shuttle vector for the genetic manipulation of mycobacterial species.