Novel Type II Heat-Labile Enterotoxin Produced by Human Disease Outbreak Isolates of Escherichia coli Is a Member of the LT-IIa Family and Should Not Form a New Group: Clarification of Nomenclature and Precedent

Dear Editor, I was very interested to read the report by Ishimaru et al. [1] describing outbreaks of human disease caused by type II (LT-II-producing) enterotoxigenic Escherichia coli (ETEC). While type II ETEC strains have been isolated from both symptomatic and healthy individuals [2], they have not previously been documented to cause outbreaks in humans, and the majority were isolated from animals [3]. This report is clear and convincing but does, however, have several issues that need addressing. First, the authors use nonstandard nomenclature for the 2 types of heat-labile enterotoxins that are, by consensus, referred to as LT-I and LT-II [4], not LT1 and LT2. In fact, LT1 and LT2 have been used to denote specific lineages of LT-I toxins [5]. Second, I disagree with the assertion that the novel LT-II toxin “formed a branch distinct from known elt2 variants” (that was designated LT2d) as, while the LT-II toxin is clearly novel, my analysis shows that it is closely related to LT-IIa and should be described as an LT-IIa variant. Third, in a previous publication cited in the report [6], I noted the existence of a novel LT-II locus in an avian E. coli genome (B088; NZ_GG749133), distinct from all LT-IIa, b, or c variants known at that time (Figure 1C). This should take precedence for the LT-IId designation. A, Sequence comparison (ClustalW alignment) of the A-B gene junctions in LT2d and LT-IIa. DNA sequences are shown in lowercase, translation in uppercase (LT2d, above, LT-IIa, below DNA sequence). * and × denote termination codons. Identities are shown by a period, differences highlighted with a teal background; single bp deletion (-) is highlighted with a red box. “Orig RF” denotes original reading frame for the LT2d A subunit sequence, now degenerated. The coding frame for B subunits has been maintained. B, Neighbor-joining bootstrap analysis of evolutionary relationships of ClustalW aligned DNA sequences for all LT-II operons (start codon of A to stop codon of B genes), with LT-I as an outlier. The optimal tree with the sum of branch length = 1.93925762 is shown. The percentages of replicate trees in which the associated toxin operons clustered together in the bootstrap test (500 replicates) are shown next to the branches [8]. The tree is drawn to scale (bar shows 0.05 base substitutions per site), with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method [9]. The analysis involved 14 nucleotide sequences, including an additional novel LT-II toxin, LT-IIe, identified in the VREC0623 genome (NZ_UDBM01000062). All positions containing gaps and missing data were eliminated. There were a total of 1023 positions in the final data set. Evolutionary analyses were conducted in MEGA7 [10] on ClustalW aligned sequences output from Clone Manager Professional, version 9.51 (SciEd software, Denver, CO, USA). Red boxes group highly related toxins into families. C, ClustalW amino-acid alignment of all LT-II B polypeptides to LT-IIa B. Amino acids are shown in single-letter lowercase, with identities to LT-IIa B shown by a period and differences highlighted with a teal background. The black box encloses signal sequences, and red boxes show regions of extreme conservation (90%–98% identity) of LT2d (LT-IIav1) and LT-IIav2 B polypeptides, with LT-IIa B encompassing all but 6 of the 100 residues of the mature B subunit. For clarity, only 3 of the 6 known LTIIc variants (representing the 3 LTIIc subbranches (B)) are shown. One unique detail regarding LT2d is that its A subunit is 7 residues shorter than all others (Figure 1A) and lacks a recognizable R/KDEL sequence at its carboxyl terminus, which is important for full toxicity and intracellular trafficking [7]. Nevertheless, this novel LT-II toxin is clearly able to function both in vitro and in vivo. However, a global alignment of the complete LT2d locus with LT-IIa reveals that this is caused by a single bp deletion in the C-terminal coding region of LT2d A, changing the reading frame (Figure 1A); a new stop codon terminates the A polypeptide, absent which the A gene would be translationally fused to the B gene. This is insufficient to designate this toxin as a new LT-II subgroup. The report showed neighbor-joining trees for the A and B genes, separately, that produced trees with different branch points, which was used to claim that LT2d forms a branch distinct from all other LT-II toxins. In analyzing the ancestry of these toxins, we must, however, consider the locus as a whole—both the A and B genes together—as this is the evolutionary unit upon which selection acts. A neighbor-joining analysis of complete operons (Figure 1B) clearly shows that LT2d, and a further novel variant I found in the NCTC10082 genome (NZ_ UFZA01), are more closely related to each other and to LT-IIa than to any other LT-II. Comparing B polypeptides, which are the most diverse subunit, LT2d and the NCTC10082 toxin each have 2 lengthy regions (of 31–50 residues, together encompassing 94% of the mature B subunits) with 90%–98% identity to LTIIa B (Figure 1C). This confirms that they are descended from LT-IIa. This degree of identity and conservation is seen elsewhere only between toxin variants that are grouped into the LT-IIc family. I propose LT2d (and other homologous loci, eg, NCTC9109, NZ_UGDU01) (Figure 1B) be renamed LT-IIav1 (LT-IIa variant 1), and the toxin from NCTC10082 designated LT-IIav2, as novel members of the LT-IIa family. A 500-repetition bootstrap analysis supports this conclusion, as it shows ≥95% confidence in all nodes (100% for the LTIIc and new LT-IIa families) except for the LT-IIa family-b/d split (81%) and internal LT-IIc family nodes (Figure 1B). While the degree of difference (average changes per bp) for designating a new subgroup is somewhat arbitrary, these novel variants clearly group with LT-IIa at the operon level and have highly similar B subunits. In conclusion, this is a very interesting report documenting a novel LT-II toxin that is more accurately described as an LT-IIa variant than a novel LT-II group; the LT2d designation should be replaced with LT-IIav1, with LT-IId being reserved for the B088 toxin locus. I agree with their conclusion that “more attention should be paid to infections by E. coli strains producing LT(-II) with colonization factors”; this should be expanded to incude all LT-II-encoding isolates, as it is clear from my analysis of other genomes that the LT-II family is even more diverse than currently accepted (awaiting demonstration of toxicity by genome-identified isolates). This study suggests that acquisition of a human colonization factor (plasmid) may be all that is required to confer on animal and environmental type II ETEC isolates the ability to colonize and produce disease in humans. Potential conflicts of interest. Author declares no conflicts of interest. The author has submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.