Impact of a human gut microbe on Vibrio cholerae host colonization through biofilm enhancement
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
In this work, the authors study the previously reported positive association between the presence of the gut bacterium Paracoccus aminovorans and Vibrio cholerae during infection. They describe and image dual species-biofilm formed in vitro as well as enhanced V. cholerae gut colonization in the presence of P. aminovorans in a neonatal mouse model. Collectively, the authors conclude that P. aminovorans enhances biofilm formation by Vc, which could explain the increased susceptibility of P. aminovorans-containing humans in cholera endemic areas.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Microbiology and Infectious Disease (eLife)
Abstract
Recent studies indicate that the human intestinal microbiota could impact the outcome of infection by Vibrio cholerae, the etiological agent of the diarrheal disease cholera. A commensal bacterium, Paracoccus aminovorans, was previously identified in high abundance in stool collected from individuals infected with V. cholerae when compared to stool from uninfected persons. However, if and how P. aminovorans interacts with V. cholerae has not been experimentally determined; moreover, whether any association between this bacterium alters the behaviors of V. cholerae to affect the disease outcome is unclear. Here, we show that P. aminovorans and V. cholerae together form dual-species biofilm structure at the air–liquid interface, with previously uncharacterized novel features. Importantly, the presence of P. aminovorans within the murine small intestine enhances V. cholerae colonization in the same niche that is dependent on the Vibrio exopolysaccharide and other major components of mature V. cholerae biofilm. These studies illustrate that multispecies biofilm formation is a plausible mechanism used by a gut microbe to increase the virulence of the pathogen, and this interaction may alter outcomes in enteric infections.
Article activity feed
-
-
Author Response:
Reviewer #1:
In this study, Barrasso et. al., investigate the previously reported positive association between the commensal P. aminovorans and V. cholerae in the human gut during infection. The authors find that P. aminovorans and V. cholerae interact in vitro to form a dual-biofilm. Using a suckling mouse model of infection, the authors also demonstrate that V. cholerae gut colonization is enhanced in the presence of P. aminovorans, and that this colonization enhancement depends on the ability of V. cholerae to produce biofilm exopolysaccharide and biofilm proteins. Overall, the experiments are well-performed and the findings are interesting. My major comment is that the authors should perform some further analysis to demonstrate a definitive causal relationship between the abundance of P. aminovorans and V. …
Author Response:
Reviewer #1:
In this study, Barrasso et. al., investigate the previously reported positive association between the commensal P. aminovorans and V. cholerae in the human gut during infection. The authors find that P. aminovorans and V. cholerae interact in vitro to form a dual-biofilm. Using a suckling mouse model of infection, the authors also demonstrate that V. cholerae gut colonization is enhanced in the presence of P. aminovorans, and that this colonization enhancement depends on the ability of V. cholerae to produce biofilm exopolysaccharide and biofilm proteins. Overall, the experiments are well-performed and the findings are interesting. My major comment is that the authors should perform some further analysis to demonstrate a definitive causal relationship between the abundance of P. aminovorans and V. cholerae colonization, which would help to strengthen their conclusions.
- The authors find a positive correlation between the abundance of P. aminovorans and V. cholerae infection by the observation that 6 of 22 infected individuals harbored detectable levels of P. aminovorans in rectal swabs, compared to only 2 of the 36 uninfected individuals (Figure 1). The authors then go on to demonstrate that there is an increase in the colonization of V. cholerae (CFU) in the presence of P. aminovorans in the suckling mouse model on infection (Figure 2B and Figure 2C). The author's conclusions would be strengthened by demonstrating a positive association between the presence of P. aminovorans and the abundance of V. cholerae in the human samples (from Figure 1). The authors demonstrate that the presence of P. aminovorans is associated with the number of people infected with V. cholerae, but not that the presence of P. aminovorans also leads to higher levels of V. cholerae in the 6 of infected individuals harboring detectable levels of P. aminovorans, compared to the infected individuals that did not. This additional analysis would help to solidify the authors conclusions that the presence of P. aminovorans enhances the colonization of V. cholerae during infection.
In the prior study in which the association between P. aminovorans and V. cholerae infection was first noted (Midani F et al, JID,, July13;218[4] :645-653), among the 4,181 different operational taxonomic units ([OTU], a taxonomic grouping based roughly on species-level identification) identified in the stool of persons infected with V. cholerae, Pa was among the top 5 OTUs found to be most abundant during infection in a machine learning analysis. This analytic method provided additional resolution about the correlative relationships between gut microbes and clinical outcomes beyond that of community structure-based analyses (Midani 2018). We have added new Supplementary File 1 which contains the full data (raw and normalized abundance) with counts in persons with and without Pa found in the stool from the Midani 2018 study. Total bacterial counts in the stool of each group did not differ, and the average Vc OTUs were higher in the group with Pa in the stool; however, this difference was not statistically significant (Supplemental File 1), which may have been due to an underpowered comparison. The wide range of Vc abundance in the human samples reflects the cross-sectional nature of this human data in that study participants are likely to be at different stages of colonization or infection at the time of sampling (compared to mice who are sampled at the same time since infection). The limitations of V. cholerae stool culture for diagnosis of infection are evident in Supplemental File 1, in which 16S detection of Vc and culture positivity were discordant in some samples (see column 3, Vc infection determination, in Supplemental File 1, all previously published data). Notably, stool culture is the gold standard for diagnosing V. cholerae infection. Rationale and additional information about these classifications and the machine learning analysis methods are included in the prior manuscript (Midani 2018). In summary, we feel that our human data from the prior study are not of sufficient in sample size to fully resolve the question asked by the reviewer, and we have added the statistical testing of Vc counts in Pa- infected and Pa-uninfected humans in our results section and to Supplemental File 1. We have also included these results in the revised manuscript. We have also included the raw and normalized abundances data in Supplementary File 1.
- It's surprising that the relative abundance Proteobacteria does not change much after the introduction of >10^6 CFU of P. aminovorans, which would be expected to represent a significant proportion of the abundance of the total bacteria in the small intestine (Supplemental Figure 1). It would be important for the authors to determine whether the addition of P. aminovorans and not the displacement of other members of the Proteobacteria Phylum leads to the increased V. cholerae CFU in Figure 2B and Figure 2C.
To show with more resolution the impact of Pa on the gut microbiota of the mouse we have added a Panel C to Figure 2-figure supplement 1 Panel C (original Supplemental Figure 1) showing order-level abundance data within the Proteobacteria phylum (to which Pa belongs). Differences in the two groups were not found to be significant with FDR-adjusted significance testing at multiple taxonomic levels. As in humans, it may be the case that a small amount of Pa is sufficient to impact Vc colonization, and it could be that only small amounts of Pa remain after inoculation.
- Expression analysis should be performed to determine whether there is an increase in virulence factor expression in V. cholerae in the presence of P. aminovorans. This important given that previous studies have demonstrated that biofilm growth leads to the upregulation of V. cholerae virulence factors, which may enhance colonization.
We have added new Figure 7-figure supplement 1 showing the qRT-PCR results for measuring the relative transcript levels of ctxA and tcpA in Vc monoculture and Vc-Pa co-culture under two different culture conditions. First, we compared virulence gene expression under the AKI growth conditions (Iwanaga et al, Microbiology and immunology, 30(11), 1075–1083) because El Tor biotype Vc does not express virulence genes optimally under standard lab growth conditions (DiRita et al, PNAS, 93(15):7991-5). Under the AKI conditions, we observed a slight increase of the transcript levels of tcpA, but not ctxA, in Vc cocultured with Pa. It should be noted that, in addition to the use of a special medium, a biphasic growth (static growth followed by shaking for extended period) is needed for inducing virulence gene expression in AKI. We reasoned that such growth conditions did not allow Vc to interact sufficiently with Pa to form a robust biofilm, likely preventing a strong induction of virulence gene expression in the AKI coculture.
We also measured virulence gene expression in regular LB medium under static growth conditions (similar to the conditions used to grow the coculture biofilms). As discussed above, we noted that under these growth conditions the relative transcript levels of these virulence genes are low (e.g., compared to the relative transcript levels of vpsL), suggesting this is not an ideal condition for virulence gene expression comparison in mono- and co-culture. Nonetheless, we found that ctxA and tcpA transcript levels are slightly higher in the static LB coculture (although only ctxA comparison is statistically significant).
Reviewer #3:
The presence of the organism Paracoccus aminovorans in stool was previously shown to correlate with susceptibility of humans to infection with V. cholerae and to enhance agglutination and growth of V. cholerae. In this manuscript, the authors use a neonatal mouse model as well as in vitro models to demonstrate that the association between Paracoccus aminovorans and V. cholerae occurs in a VPS-dependent biofilm and enhances colonization of the neonatal mouse intestine.
The strengths of this manuscript:
- Examination of P. aminovorans-V. cholerae interaction in the small intestine of the neonatal mouse model. V. cholerae colonizes the terminal ileum yet most of the human microbiota studies examine stool, which is unlikely to be representative of the terminal ileum. In addition, adult models of infection such as the gnotobiotic or antibiotic-treated mouse display colonic but not true ileal colonization. Furthermore, this colonization is not dependent on the V. cholerae toxin co-regulated pilus, which is necessary for human infection. In fact, flow in the colon is slow enough to allow growth without true attachment to the surface. This may explain why, as the authors note, the diarrhea of cholera clears most of the microbiota from wtool samples. Therefore, stool exiting the colon and the adult mouse are not ideal for studying the interaction between the microbiota and V. cholerae during infection. By using the neonatal mouse, the authors choose a host compartment that is relevant to human disease. The findings of the authors that P. aminovorans improves V. cholerae colonization of the small intestine are very convincing.
- Use of microscopy to detail the distribution of the two organisms in culture. Imaging clearly demonstrates subdomains of the biofilm that contain mixtures of P. aminovorans and V. cholerae.
The weaknesses of this manuscript:
- Specific markers for VPS exopolysaccharide and P. aminovorans are not used: The authors conclude that V. cholerae increases VPS synthesis in response to P. aminovorans based on increased WGA staining in regions of biofilms where P. aminovorans is concentrated. One concern is that WGA is not a specific marker for VPS. It adheres to GlcNAC residues, which could also be present in an extracellular polysaccharide synthesized by P. aminovorans.
In response to the reviewer’s question, we added negative controls (new Figure 4-figure supplement 1) showing that Pa cells along or Vc ΔvpsL mutant does not show WGA staining. While it is true that WGA stains GlcNAC residues, for Gram-negative bacterial cells including Vc and Pa, the WGA lectin molecules do not pass through the outer membrane. Only when the outer membrane is significantly impaired will one see strong WGA signal. Indeed, we always observe dead cells with strong WGA signal but those are easily distinguished from the VPS signal. We have updated Figure 6A-B accordingly to better show the local distribution of VPS.
Furthermore, the authors image biofilms that include neon-green-expressing V. cholerae and unlabeled P. aminovorans by staining with FM4-64. Regions of the biofilm with predominantly FM4-64 staining are presumed to have greater numbers of P. aminovorans. This is more convincing because the shape of these cells is coccoid as might be expected for P. aminovorans. However, this is not a feature that provides specificity. Because the authors also indicate that these clusters of Pa are surrounded by V. cholerae¬-synthesized VPS, it is important to definitively identify these cells as Pa.
We thank the reviewer for raising this point. In the new Figure 6A-B, we have provided clearer zoomed-in images in which Pa cells can be clearly distinguished from Vc cells by BOTH the absence of SCFP3A signal AND the characteristic cocci shape.
- There is no examination of activation of VPS synthesis or other virulence factors at the transcriptional level. The authors conclude that VPS synthesis is activated by WGA staining but do not provide additional data to show whether this activation occurs at the transcriptional or post-transcriptional level. If transcription activation of vps genes were observed, this would bolster the WGA staining result.
In response to the reviewer’s request, we have obtained a strain harboring Pvpsl-mNeonGreen and we have repeated the pellicle imaging in the updated Figure 6C-D. Interestingly, subpopulations of Vc cells have elevated vpsL expression when co-cultured with Pa. In addition, we have performed qRT-PCR experiments to show that indeed the relative transcript levels of vpsL is higher in the co-culture (Figure 5).
-
Evaluation Summary:
In this work, the authors study the previously reported positive association between the presence of the gut bacterium Paracoccus aminovorans and Vibrio cholerae during infection. They describe and image dual species-biofilm formed in vitro as well as enhanced V. cholerae gut colonization in the presence of P. aminovorans in a neonatal mouse model. Collectively, the authors conclude that P. aminovorans enhances biofilm formation by Vc, which could explain the increased susceptibility of P. aminovorans-containing humans in cholera endemic areas.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
Reviewer #1 (Public Review):
In this study, Barrasso et. al., investigate the previously reported positive association between the commensal P. aminovorans and V. cholerae in the human gut during infection. The authors find that P. aminovorans and V. cholerae interact in vitro to form a dual-biofilm. Using a suckling mouse model of infection, the authors also demonstrate that V. cholerae gut colonization is enhanced in the presence of P. aminovorans, and that this colonization enhancement depends on the ability of V. cholerae to produce biofilm exopolysaccharide and biofilm proteins. Overall, the experiments are well-performed and the findings are interesting. My major comment is that the authors should perform some further analysis to demonstrate a definitive causal relationship between the abundance of P. aminovorans and V. cholerae …
Reviewer #1 (Public Review):
In this study, Barrasso et. al., investigate the previously reported positive association between the commensal P. aminovorans and V. cholerae in the human gut during infection. The authors find that P. aminovorans and V. cholerae interact in vitro to form a dual-biofilm. Using a suckling mouse model of infection, the authors also demonstrate that V. cholerae gut colonization is enhanced in the presence of P. aminovorans, and that this colonization enhancement depends on the ability of V. cholerae to produce biofilm exopolysaccharide and biofilm proteins. Overall, the experiments are well-performed and the findings are interesting. My major comment is that the authors should perform some further analysis to demonstrate a definitive causal relationship between the abundance of P. aminovorans and V. cholerae colonization, which would help to strengthen their conclusions.
The authors find a positive correlation between the abundance of P. aminovorans and V. cholerae infection by the observation that 6 of 22 infected individuals harbored detectable levels of P. aminovorans in rectal swabs, compared to only 2 of the 36 uninfected individuals (Figure 1). The authors then go on to demonstrate that there is an increase in the colonization of V. cholerae (CFU) in the presence of P. aminovorans in the suckling mouse model on infection (Figure 2B and Figure 2C). The author's conclusions would be strengthened by demonstrating a positive association between the presence of P. aminovorans and the abundance of V. cholerae in the human samples (from Figure 1). The authors demonstrate that the presence of P. aminovorans is associated with the number of people infected with V. cholerae, but not that the presence of P. aminovorans also leads to higher levels of V. cholerae in the 6 of infected individuals harboring detectable levels of P. aminovorans, compared to the infected individuals that did not. This additional analysis would help to solidify the authors conclusions that the presence of P. aminovorans enhances the colonization of V. cholerae during infection.
It's surprising that the relative abundance Proteobacteria does not change much after the introduction of >10^6 CFU of P. aminovorans, which would be expected to represent a significant proportion of the abundance of the total bacteria in the small intestine (Supplemental Figure 1). It would be important for the authors to determine whether the addition of P. aminovorans and not the displacement of other members of the Proteobacteria Phylum leads to the increased V. cholerae CFU in Figure 2B and Figure 2C.
Expression analysis should be performed to determine whether there is an increase in virulence factor expression in V. cholerae in the presence of P. aminovorans. This important given that previous studies have demonstrated that biofilm growth leads to the upregulation of V. cholerae virulence factors, which may enhance colonization.
-
Reviewer #2 (Public Review):
In the manuscript "Impact of a human gut microbe on Vibrio cholerae host colonization through biofilm enhancement" the authors studied the interaction between the human associated microbiota member Paracoccus aminovorans and the human pathogen Vibrio cholerae. A previous study, where authors from the same lab analyzed microbiota composition of individuals infect and non-infected with V. cholerae led to the hypothesis that P. aminovorans could increase colonization of Vibrio cholerae. In the present manuscript the authors used a murine model to test this hypothesis and in vitro laboratory cultures to identify possible mechanisms involved in the interaction between P. aminovorans and V. cholerae.
They could show that in a murine model P. aminovorans indeed increases the colonization levels of V. cholerae.
With …
Reviewer #2 (Public Review):
In the manuscript "Impact of a human gut microbe on Vibrio cholerae host colonization through biofilm enhancement" the authors studied the interaction between the human associated microbiota member Paracoccus aminovorans and the human pathogen Vibrio cholerae. A previous study, where authors from the same lab analyzed microbiota composition of individuals infect and non-infected with V. cholerae led to the hypothesis that P. aminovorans could increase colonization of Vibrio cholerae. In the present manuscript the authors used a murine model to test this hypothesis and in vitro laboratory cultures to identify possible mechanisms involved in the interaction between P. aminovorans and V. cholerae.
They could show that in a murine model P. aminovorans indeed increases the colonization levels of V. cholerae.
With laboratory cultures they also showed that P. aminovorans increases the ability of V. cholerae to form biofilms. Importantly, the P. aminovorans dependent increase in V. cholerae biomass was specific to V. cholerae cells in biofilm mode, and not a general increase in growth Because an increase in growth was observed for cells in the biofilms but not in planktonic conditions. Moreover, components required for biofilm formations, such as Vibrio exopolysaccharides, were required for the observed induction of P. aminovorans increase in V. cholerae biofilm formation and in gut colonization. The authors propose that P. aminovorans induces biofilm formation in V. cholerae and that promotes V. cholerae gut colonization.
Overall, the study described here is well written and the conclusions are generally well supported by the data.
Importantly, this study shows that indeed there is a direct interaction between P. aminovorans and V. cholearae and thus supporting the hypothesis that the observed increase in V. cholerae colonization in the presence of P. aminovorans is the result of this direct interaction. The results from in vitro studies shown here demonstrating the formation of dual species biofilms with these two organisms, in addition to the observation that the P. aminovorans mediated increase in gut colonization of V. cholerae is no longer observed when mutants incapable of forming biofilms are used, provide strong support to the proposal that the formation of dual species biofilms are a likely mechanism explaining the ability of P. aminovorans to enhance V. cholerae virulence. In future work it will be interesting to see the formation P. aminovorans and V. cholerae dual species biofilms directly in the gut, to obtain direct support for the role of the biofilms in the gut.
More broadly, this work highlights the importance of looking at the interactions between members of the microbiota and pathogens to understand different host susceptibility to infection outcomes. Other studies have looked at how members of the microbiota can decrease pathogen intestinal colonization. Here, an increase in pathogen colonization is demonstrated by an interaction with a specific microbiota member. Identification of these interactions can be important to identify infection susceptibilities, but also to develop strategies that prevent these interactions as new ways to decrease infections.
-
Reviewer #3 (Public Review):
The presence of the organism Paracoccus aminovorans in stool was previously shown to correlate with susceptibility of humans to infection with V. cholerae and to enhance agglutination and growth of V. cholerae. In this manuscript, the authors use a neonatal mouse model as well as in vitro models to demonstrate that the association between Paracoccus aminovorans and V. cholerae occurs in a VPS-dependent biofilm and enhances colonization of the neonatal mouse intestine.
The strengths of this manuscript:
Examination of P. aminovorans-V. cholerae interaction in the small intestine of the neonatal mouse model. V. cholerae colonizes the terminal ileum yet most of the human microbiota studies examine stool, which is unlikely to be representative of the terminal ileum. In addition, adult models of infection such as …
Reviewer #3 (Public Review):
The presence of the organism Paracoccus aminovorans in stool was previously shown to correlate with susceptibility of humans to infection with V. cholerae and to enhance agglutination and growth of V. cholerae. In this manuscript, the authors use a neonatal mouse model as well as in vitro models to demonstrate that the association between Paracoccus aminovorans and V. cholerae occurs in a VPS-dependent biofilm and enhances colonization of the neonatal mouse intestine.
The strengths of this manuscript:
Examination of P. aminovorans-V. cholerae interaction in the small intestine of the neonatal mouse model. V. cholerae colonizes the terminal ileum yet most of the human microbiota studies examine stool, which is unlikely to be representative of the terminal ileum. In addition, adult models of infection such as the gnotobiotic or antibiotic-treated mouse display colonic but not true ileal colonization. Furthermore, this colonization is not dependent on the V. cholerae toxin co-regulated pilus, which is necessary for human infection. In fact, flow in the colon is slow enough to allow growth without true attachment to the surface. This may explain why, as the authors note, the diarrhea of cholera clears most of the microbiota from wtool samples. Therefore, stool exiting the colon and the adult mouse are not ideal for studying the interaction between the microbiota and V. cholerae during infection. By using the neonatal mouse, the authors choose a host compartment that is relevant to human disease. The findings of the authors that P. aminovorans improves V. cholerae colonization of the small intestine are very convincing.
Use of microscopy to detail the distribution of the two organisms in culture. Imaging clearly demonstrates subdomains of the biofilm that contain mixtures of P. aminovorans and V. cholerae.
The weaknesses of this manuscript:
- Specific markers for VPS exopolysaccharide and P. aminovorans are not used: The authors conclude that V. cholerae increases VPS synthesis in response to P. aminovorans based on increased WGA staining in regions of biofilms where P. aminovorans is concentrated. One concern is that WGA is not a specific marker for VPS. It adheres to GlcNAC residues, which could also be present in an extracellular polysaccharide synthesized by P. aminovorans.
Furthermore, the authors image biofilms that include neon-green-expressing V. cholerae and unlabeled P. aminovorans by staining with FM4-64. Regions of the biofilm with predominantly FM4-64 staining are presumed to have greater numbers of P. aminovorans. This is more convincing because the shape of these cells is coccoid as might be expected for P. aminovorans. However, this is not a feature that provides specificity. Because the authors also indicate that these clusters of Pa are surrounded by V. cholerae¬-synthesized VPS, it is important to definitively identify these cells as Pa.
- There is no examination of activation of VPS synthesis or other virulence factors at the transcriptional level. The authors conclude that VPS synthesis is activated by WGA staining but do not provide additional data to show whether this activation occurs at the transcriptional or post-transcriptional level. If transcription activation of vps genes were observed, this would bolster the WGA staining result.
-
-