In this study, we sought to understand how diversity in the gene encoding the V. cholerae
MARTX toxin might affect the function of the toxin both in clinical isolates and in the environment. Recent studies have shown that the composite MARTX toxin genes can be altered in V. vulnificus
by homologous recombination to acquire new effector domains (24
), and we were interested if similar variation occurred in V. cholerae
. For genomic analysis, it was first necessary to reconcile discrepancies in the annotation of the rtxA
gene of the representative strain N16961. We showed experimentally that the correct start site is the ATG downstream of rtxC
, although some activity may arise from the upstream GTG. Other Vibrio
spp. have been identified to have rtxA
genes for MARTX toxins (20
). Analysis of the upstream sequences for rtxA
genes from V. vulnificus
), V. anguillarum
), V. splendidus
), and V. caribbenthicus
(NZ_AEIU01000000) reveal that these each contain a conserved trinucleotide GTG and ATG start site as well as a conserved TAA stop codon for rtxC
. However, the intervening sequences vary in comparison to N16961. Most notably, in no cases would use of the GTG as a start site introduce the appropriate frame of translation for the MARTX toxin, indicating that in all Vibrio
spp., the start site will be conserved with the ATG of V. cholerae
, as mapped in this study.
Using the correct 13,638-bp gene, we examined diversity of this gene in 71 V. cholerae
strains for which the entire gene was represented in a sequenced genome supplemented by available SNP data from recent whole-genome phylogeny (2
). Whole-genome studies have revealed that the El Tor O1 lineage differs from N16961, with a calculated rate of only 3.3 SNPs/year (2
). Not surprisingly, given the global conservation of these strains, few SNPs were identified in the rtxA
gene despite the 100-year history of available data. In total, only 9 rtxA
alleles were identified across all El Tor O1, O139, atypical, and altered El Tor strains.
All of the El Tor seventh pandemic strains shared an adenosine nucleoside at rtxA
nt 8731, distinct from a guanosine present in pre-seventh pandemic strains, other El Tor-like lineage strains from the United States Gulf Coast and Australia, and the O37 strain V52. The nucleoside change results in a codon change from glycine in prepandemic strains to serine in pandemic strains within the RID effector domain. Notably, among other MARTX toxins with an RID, a Gly at this position is most prevalent (38
). This finding of a conservative mutation unique to the El Tor seventh pandemic MARTX RID is consistent with the El Tor strains, representing a clonal expansion from a single predecessor at some point in the 1950s diverging away from other El Tor-like lineages (2
). All together, the strong conservation of this gene, with only 1 to 6 SNPs across 100 years and worldwide geography, reinforces that these strains are closely related evolutionarily and distinct from other V. cholerae
The conservation of the rtxA
sequence in the El Tor O1 and closely related strains is in stark contrast to the extensive variation in the non-El Tor group, whether the strain was of environmental or clinical origin. The database for these strains is more limited, although each of the 12 isolates had a unique rtxA
gene that differed from the reference strain by between 176 and 321 nt, or 1 to 2%. This variation matches the calculated 2% rate of change for whole-genome variation of 3 environmental isolates recently reported (8
). This dramatic variation was not expected, because in a recent multi-virulence locus sequence typing analysis, including both O1 and non-O1 strains, non-O139 isolates showed limited variation in the rtxA
). Interestingly, this analysis was focused to 332 bp from nt 1339 to 1671, and assessment of the SNP map for our analysis showed limited SNPs in this region (). Conservation in this region is in contrast to the spacer region between the B repeats and the ACD effector domain where extensive variation occurred, indicating that conservation of rtxA
, as previously reported (39
), was biased by the region analyzed and does not reflect total diversity across the large gene. Many of the SNPs in the genomes seemed to arise in clusters across several genomes, suggesting that variation in this gene arises extensively from homologous recombination and the regions amenable to homologous recombination may be defined in some way by DNA structure or nucleotide sequence. In all, in contrast to the lack of variation within the El Tor group, there is a large capacity for the rtxA
gene to vary in the environmental strains.
Two genes were identified that are new toxin variants due to a change in the central portion of rtxA that encodes the effectors. These changes converted one of the toxins to a potential adenylate cyclase toxin and another to a putative actin ADP-ribosylating toxin. Due to extensive nucleotide diversity in the conserved regions, the site of recombination events could not be identified. The consequences of these genetic changes will need to be further explored in the future, as will whether these changes affected the biological niche these strains occupy.
The most clinically relevant change in the rtxA toxin gene was one SNP that emerged within the currently circulating strains known as the altered El Tor strain. This SNP inactivated the function of the MARTX toxin by introducing a premature stop codon that would result in a protein truncated by 12 amino acids, probably disrupting the C-terminal secretion signal. Strains 2011EL-1098, 2009EV-1131, and 2011EL-1132 were deleted from analysis for out-of-frame mutations, but if these mutations could be validated, they would indicate that this group of strains is accumulating additional null mutations in rtxA, generating a pseudogene that could not be reversed by a single nucleotide reversion.
Subsequent to the emergence of rtxA
allele 4, another significant mutation apparently arose within the rtxA
-null background in which a point mutation in ctxB
created a CtxB with a never-before-observed Asn at aa 20. As no strains were identified in which this ctxB7
allele occurred with an intact rtxA
gene, it is suggested that ctxB7
arose within the rtxA
-null background. This genetic cluster is represented by 27 different sequenced strains and extensive additional WGS analysis, demonstrating that strains with both mutations have been transmitted globally, reaching India, Nepal, Cameroon, Haiti, and the Dominican Republic (2
). The data also indicate that in addition to comprising a clonal cluster that may have originated in India (7
), these strains have a distinct virulence factor profile from other El Tor strains (6
). Therefore, it is necessary when categorizing the altered El Tor to distinguish not only the ctxB
allele but also the rtxA
allele to determine if the strain produces an active MARTX toxin. It is yet to be determined which of these mutations, if either, can account for the increased virulence of these strains in humans (6
), although 2010EL-1786 is not statistically different in virulence from typical El Tor strains in infant mice (12
) or in adult mice (our unpublished findings).
Interestingly, the classical strains responsible for historical cholera pandemics are also noted for a naturally occurring deletion that removes >7 kb of the rtx
locus, deactivating the MARTX toxin (19
). The question then is, as strains become more virulent and adapted for human-to-human passage, why do they deactivate MARTX? The toxin is very large and may be detrimental to growth due to energy expenditure reducing rapid growth necessary for increased dissemination. If the toxin has a role in the environment, it may no longer be necessary if the strain acquires the ability to transfer host to host, making circulation through the environment less essential. The toxin has been shown in mice to have a function in virulence by promoting colonization of the intestine through compromising the innate immune system during early colonization (40
). However, it has been shown that the toxin is fully redundant in function with hemolysin, a pore-forming toxin (42
). Strain 2010EL-1786, the representative strain used in this study, has been reported to be hemolytic, similar to other El Tor strains (12
), and we independently confirmed this result (data not shown). Thus, the loss of function of the rtxA
gene may be circumvented by the action of secreted hemolysin.
Yet, classical strains are nonhemolytic in addition to not producing a MARTX toxin. It has been postulated that innate immune evasion is a key function of CT, in addition to its ability to induce enterocyte secretion (43
). It is possible that in the context of excess production of classical-type CT, as occurs in classical strains and the altered El Tor strains now circulating, neither MARTX nor hemolysin is required for innate immune evasion, their function being replaced by the immunomodulatory function of the classical form of CtxB.
Overall, we have found that the MARTX toxin in general has been highly conserved in V. cholerae, but when variants do arise, they can dramatically alter protein function and possibly overall process of pathogenesis. Thus, while it is clear that analysis of whole genomes can provide phylogenetic evidence of how strains are evolving over time, detailed analysis of individual genes is still necessary to reveal how small changes in nucleotide sequence can dramatically affect protein function, and these mutations may contribute to increased fitness and global spread.