Vibrio cholerae article comments:

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J Bacteriol. 2017
Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae
Yamamoto S, Ohnishi M

The model for the chitin catabolic cascade indicates GlcNAc oligomers are degraded in the periplasm to ABC-transported (GlcNAc)2 and PTS-transported GlcNAc (Figure 5 in Park JK, 2002 for the original model).  The authors used (GlcNAc)4 and, because Enzyme IIAGlc was largely phosphorylated in the presence of (GlcNAc)4, proposed there was "some mechanism for which chitin oligosaccharides escape from degradation into GlcNAc in the periplasmic space".  If such mechanism occurs under the authors’ experimental conditions, it precludes any major PTS-transport effects on the chitin cascade, i.e., via dephosphorylation of Enzyme IIAGlc during GlcNAc transport.

Working with Vibrio furnissii, Keyhani NO, 1996 argued, "since (GlcNAc)2 is an important inducer in the cascade, it must resist hydrolysis in the periplasm", and further provided an explanation for the stability of (GlcNAc)2 in the periplasm, particularly in sea water.  It may well be that the rapid catabolism of (GlcNAc)2 is 'free' from any PTS control, and as such the cAMP necessary for the chitin cascade is provided.

This comment by Martine Crasnier-Mednansky (martine [at] minst [dot] org) was originally posted on Sep 18, 2017 at PubMed Commons

Environ Microbiol. 2017a
Systematic genetic dissection of chitin degradation and uptake in Vibrio cholerae
Hayes CA, Dalia TN, Dalia AB

The authors really should not write in legend of Figure 7, "Our results indicate that for robust growth on chitin, the transporters responsible for uptake of [ABC-transported] chitobiose and [PTS-transported] GlcNAc play the largest role".  In fact, data in Figure 6A indicate lack of either one of these two transporters does not impair growth on chitin at all.  Also, the 'chitosan' PTS (PTSChs, VC1282 in the figure), cannot possibly be a major player considering there is little glucosamine in chitin.  Moreover, PTS transport is most likely inhibited by Enzyme IIA-dependent PTS transports (including PTSNag), as it was reported chs expression is positively regulated by cAMP (Berg T, 2007).

A proper question is whether or not the two transporters can be used simultaneously, as depicted in Figure 7.  Upon in vitro characterization of the periplasmic chitodextrinase activity (VCA0700 in Vibrio cholerae), Keyhani NO, 1996, proposed the ABC transport at first predominates up to a threshold concentration of (GlcNAc)n, n≥3, above which the PTS transport predominates. The two transport systems are not physiologically redundant.

This comment by Martine Crasnier-Mednansky (martine [at] minst [dot] org) was originally posted on Oct 13, 2017 at PubMed Commons

Mol Microbiol. 2017b
Systematic genetic dissection of PTS in Vibrio cholerae uncovers a novel glucose transporter and a limited role for PTS during infection of a mammalian host
Hayes CA, Dalia TN, Dalia AB

The authors’ proposal 'the PTS has a limited role during infection' and concluding remark 'PTS carbohydrates are not available and/or not utilized in the host' are both questionable.  Mondal M, 2014 established, when Vibrio cholerae colonizes the intestinal mucus, the PTS-substrate GlcNAc is utilized for growth and survival in the host intestine (upon mucin hydrolysis).

This comment by Martine Crasnier-Mednansky (martine [at] minst [dot] org) was originally posted on Jul 24, 2017 at PubMed Commons, and triggered the following discussion:

I appreciate your evaluation of the manuscript, however, I have to disagree with your comment.  The study by Mondal et al. indicates that ChiA2 can liberate GlcNAc from mucin in vitro and that it is critical for bacterial growth in vivo, however, they did not test the role for GlcNAc uptake and/or catabolism in that study.  In our manuscript, however, we demonstrate that loss of all PTS transporters (including the GlcNAc transporter) does not result in attenuation in the same infant mouse model, which is a more formal test for the role of GlcNAc transport during infection.  It is formally possible that other carbohydrate moieties are liberated via the action of ChiA2 that are required for growth of V. cholerae in vivo, however, our results would indicate that these are not translocated by the PTS.  Alternatively, the reduced virulence of the ChiA2 mutant observed in the Mondal et al. study may indicate that ChiA2 has other effects in vivo (i.e. on immune evasion, resistance to antimicrobial peptides, etc.).

I do appreciate your answer to my comment, to which I gladly reply.  First, there is prior work by Ghosh S, 2011 indicating colonization was attenuated in mutant strains that were incapable of utilizing GlcNAc, which included a nagE mutant strain.  Second, Mondal M, 2014 analyzed the products of the ChiA2 reaction and found GlcNAc was the most abundant product.  In fact, the amount of (GlcNAc)2 was found to be very low as compared to GlcNAc and (GlcNAc)3.  Therefore, it is fully legitimate to conclude the PTS substrate GlcNAc is utilized in the host by V. cholerae for growth and survival.

Int J Med Microbiol. 2016
Role of a sensor histidine kinase ChiS of Vibrio cholerae in pathogenesis
Chourashi R, Mondal M, Sinha R, Debnath A, Das S, Koley H, Chatterjee NS

This paper reinforces –perhaps validates– the authors’ previous work (Mondal M, 2014) indicating PTS-transported GlcNAc is utilized by Vibrio cholerae in the mucus layer, as further explained.  Data in Meibom KL, 2004, particularly supplemental Figure 7, clearly indicate chiA2 (VCA0027) is not upregulated by GlcNAc.  Therefore, in agreement with the present work, GlcNAc utilization by V. cholerae in the mucus may rely on the periplasmic activation of ChiS for production of ChiA2.  Because chiS mutant strains do not produce extracellular chitinases in the presence of chitin oligomers (Li X, 2004), they are unlikely to produce chitinases in the presence of mucin (the authors report ChiS is activated in the presence of mucin).  Thus, both chiA2 and chiS mutant strains may prevent colonization of the intestine because they are both unable to cause mucin hydrolysis by ChiA2 and subsequent release of GlcNAc, which, according to the authors’ original 2014 proposal, is necessary for growth and survival in the mucus.  The mucin-derived 'inducer' for ChiS activation is possibly (GlcNAc)3, as the authors reported (GlcNAc)3 is released upon mucin hydrolysis by ChiA2.

This comment by Martine Crasnier-Mednansky (martine [at] minst [dot] org) was originally posted on Oct 26, 2017 at PubMed Commons

Infect Immun. 2015
A genome wide screen reveals that Vibrio cholerae phosphoenolpyruvate phosphotransferase system (PTS) modulates virulence gene expression
Wang Q, Millet YA, Chao MC, Sasabe J, Davis BM, Waldor MK

Gene identification number VC1822 and VCA1045 relate to PTS proteins with A, B, and C domain (not just an A domain as inferred from the text).  In accordance with the 'Proposed Uniform Nomenclature' for the PTS proteins (Saier MH Jr, 1992), gene VC1822 encodes an Enzyme IIACBN/D and VCA1045 an Enzyme IICBAMtl.  It would be a great benefit for PTS researchers if the nomenclature was used beyond for example Escherichia coli.

Based on figure 6A, the authors report the CRP-cAMP complex negatively regulates the expression of tcpA in the absence of the PTS.  However, because figure 6A shows there is no significant difference in expression between the cya and the cya ptsH mutant strain or the crp and the crp ptsI mutant strain, and no significant difference in expression between the wild type and the crp or cya mutant strain, it could be concluded from figure 6A that the CRP-cAMP complex does not play a major role in the transcription of tcpA in the TCP-producing AKI medium used by the authors.  This conclusion is not supported by previous data indicating CRP-cAMP indirectly regulates negatively the expression of tcpA by inhibiting the transcription of tcpPH (Kovacikova G, 2001).  However the cAMP level varies in response to changes in carbon and energy sources.  Thus, if the level of cAMP is relatively low in the AKI medium as compared to LB medium, production of TcpA occurs.  This provides an explanation for the present observation indicating deletion of crp does not significantly affect production of TcpA (figure 6A).  It also provides an explanation why incorporation of a crp mutation in the El Tor strain C6706 allows production of TCP in LB medium (Skorupski K, 1997).

Figure 6B does not indicate that "…in the EI or Hpr mutants, intracellular cAMP concentrations were significantly higher than that in wt cells" - as stated.  In fact, intracellular cAMP concentrations are significantly higher in the cpdA mutant strains, possibly indicating a role for cAMP phosphodiesterase in regulating the cAMP levels.

This comment by Martine Crasnier-Mednansky (martine [at] minst [dot] org) was originally posted on Jul 3, 2015 at PubMed Commons

Environ Microbiol. 2012
Chitin colonization, chitin degradation and chitin-induced natural competence of Vibrio cholerae are subject to catabolite repression
Blokesch M

In the discussion, the author questions whether Vibrio cholerae chitin-induced natural competence and transformation would ever occur in nature because of two seemingly irreconcilable posits: (1) transport of the PTS-sugar GlcNAc causes dephosphorylation of Enzyme IIAGlc thus preventing cAMP synthesis, and (2) chitin-induced natural competence and transformation requires cAMP for transcriptional activation of TfoX-regulated genes.

To solve this issue, the author claims "GlcNAc might not be abundant next to the chitin surface". This is probably the case as cells reaching the chitin surface are most likely limited in carbon sources, a condition in Escherichia coli leading to a marked increase in cAMP (Botsford JL, 1978).  Under starving conditions, the cells are probably actively synthesizing chitinases and, in its natural chitin-rich habitat, chitin colonization and degradation are most likely not subject to catabolite repression, particularly at limiting concentrations of (GlcNAc)2 for growth.

As regards natural competence and transformation, the negative effect of GlcNAc transport is far from being established by the author’s data.  According to Figure 4, addition of 2 mM GlcNAc does not significantly affect transformation frequency with (GlcNAc)6 as inducer of natural transformation.  Furthermore, it appears the effect of GlcNAc on transformation frequency is independent of GlcNAc transport (Figure 2, third panel).

Meibom KL, 2005 proposed three controlling factors for natural transformation, "the presence of chitin; increasing cell density; and nutrient limitation, growth deceleration or stress".  As regards cell density, the quorum-sensing master regulator HapR is essential for natural competence, and considering utilization of GlcNAc upon growth deceleration causes an increase in cAMP, GlcNAc may actually be a good carbon source in vitro for natural transformation in the presence of chitin.

This comment by Martine Crasnier-Mednansky (martine [at] minst [dot] org) was originally posted on Dec 07, 2017 at PubMed Commons

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