A new regulatory link between chitin attachment and natural competence in Vibrio cholera

We know that Vibrio species have an affinity for chitin. We know that chitin induces competence in Vibrio. But the cellular mechanisms linking these two events elude us. Some definitions; competence is a bacterium’s ability to uptake DNA from the environment and chitin is an unimaginably common biopolymer, consisting of beta-linked N-acetylglucosamine.

Vibrio cholera is awfully fond of the chitinous exoskeleton of copepods, from which it can acquire nitrogen and carbon, in a process regulated by histidine kinase (ChiS); this protein is suppressed by periplasmic chitin binding protein (CBP) when chitin is absent. But once CBP binds chitin, ChiS gets busy; it is believed to phosphorylate (thereby deactivating) an unidentified regulator that inhibits expression of chitin degradation, uptake and utilization genes. Additionally, chitin also induces competence in V. cholera via regulation by TfoX and HapR, genes which are activated by chitin and quorum sensing, respectively.

However, the molecular dance linking chitin sensing to competence induction via TfoX activation is at present a mystery. This study has found and described a novel chitin sensing regulator that bridges this knowledge gap.

The methods do not make light reading, so I will summarize; transposon mutagenesis to give V. cholera spectinomycin resistance, bacteria grown either on chitin or chitin with kanamycin (to select for transformants), DNA isolated from both groups, DNA sequenced, sequences compared to identify the genes responsible for transformation, deletion mutants of these genes were then made, grown with tetracycline resistance plasmids, then tested for natural transformation by growing them as before but with tetracycline.

The previously known TfoS gene was found to be involved in competence induction; this gene is predicted to be a transmembrane protein with a DNA-binding site. TfoS deletion mutants did not transform in transformation inducing conditions. The position of TfoS in the regulatory cascade of natural competence was then examined; inducing overexpression of TfoX saves a TfoS mutant, so TfoS probably precedes TfoX. When grown in competence inducing and non-inducing conditions, TfoS could up regulate expression of TfoX. Using methods that I could not understand, they somehow confirmed that TfoS controls TfoX via regulation of the gene TfoR.

Afterwards, they fused a TfoR promoter to E. coli and found that it increased TfoS expression 30 fold, confirming that TfoR directly regulates TfoS.

So without chitin, TfoS cannot bind DNA. With chitin, it activates the TfoR gene, which interacts with the gene for TfoX, which activates the genes needed for binding and uptake of DNA from the environment. These genes have been previously shown to highly conserved amongst Vibrio species and likely serve similar functions. Since ChiS mutants could not grow on chitin, but TfoS mutants can, then these may be distinct chitin sensors involved in separate regulatory cascades. However another study has implicated ChiS as being part of the same regulatory cascade, so it is possible that it facilitates interaction of TfoS with chitin, or TfoR activation. Future work is proposed to be aimed at determining the binding site of TfoS and other binding targets of this protein to look for any additional roles it may have. I wonder that these cascades do during the transformation V. cholera undergoes as it becomes pathogenic in the human gut. Since HapR is quorum controlled, then surely it would be active in the gut when V. cholerae cell densities are high, so there must be some overlap between the cascades controlling the binding to the gut wall and to chitin. The human gut seems to be an ideal place for acquiring new genes, given the bacterial diversity there, but it is lacking in chitin, so it would be odd if Vibrio cholerae just turned off its competency once in a host and did not have another competence inducing system for when it is in the human gut.

Dalia, A. B., Lazinski, D. W., & Camilli, A. (2014). Identification of a Membrane-Bound Transcriptional Regulator That Links Chitin and Natural Competence in Vibrio cholerae. mBio, 5(1), e01028-13.

Vibrio parahaemolyticus type lV pili and the attachment to diatom-derived chitin

The gram-negative marine bacterium Vibrio parahaemolyticus commonly accumulates in filter feeding shellfish and can cause gastroenteritis in humans when the shellfish are ingested. V. parahaemolyticus is often isolated from coastal brackish waters where it occurs in higher abundance during the summer months. V. parahaemolyticus, like many other species within this genus, are not culturable when the environmental conditions prevent optimal growth (VBNC state).

Entering the VBNC state was previously assumed to be the main mechanism by which the bacteria persist in the environment until their attachment to plankton or other substrate has been identified as another mechanism for overwintering in the environment. The adherence to substances of many other Vibrio sp. is reliant on pili such as type IV pili and in V. vulnificus, type IV have been found to enable the bacteria to persist during seasons by binding to substrates. V. parahaemolyticus has genes for two types of IV pilins; one of which is involved in attachment to surfaces and biofilm formation (MshA), whereas the other pili enhances cell-to-cell interactions and connection one cell to another (PilA). PilA expression was found up-regulated when the bacteria were exposed to chitin rich conditions. As mentioned in recent blog posts, Vibrio sp. are often associated with chitinous zooplankton, and this study was one of the first to investigate the interactions of diatom-derived chitin and the efficiency of type IV pili adhering V. parahaemolyticus to substrates.

Bioassays were used to measure the ability of V. parahaemolyticus to form biofilms, as well as the adherence of the bacteria to the diatom Thalassiosira weissflogii. Results showed that the bacteria were able to attach themselves to the diatom-derived chitin, and additionally, adherence increased when the diatoms entered the stationary and lag phases of growth. For testing the importance of the two above mentioned genes for the type IV pili, genetic mutants were created and it was shown that mutations of the genes were less effective at attaching the bacteria to the diatoms compared to the wild types. 

In summary, this study pointed out the importance of diatoms for the attachment and persistence of V. parahaemolyticus in the environment in addition to prior findings of zooplankton adherence and the VBNC state. Results could be of significant importance for the prediction of dangerous accumulations of the human pathogen in shellfish by monitoring algal blooms as findings gave evidence for an increased accumulation of V. parahaemolyticus at a certain abundance of the algae. The adherence of the bacteria was shown to be dependent on the type IV pili and the ability to attach to chitin excreted by diatoms could be an important factor enhancing their persistence in the environment.

Frischkorn et al. (2013).Vibrio parahaemolyticus type lV pili mediate interactions with diatom-derived chitin and point to an unexplored mechanism of environmental persistence. Environmental Microbiology 15(5), pp. 1416-1427.

Probiotic Strains for Shellfish Aquaculture: Protection of Eastern Oyster, Crassostrea virginica, Larvae and Juveniles Againsl Bacterial Challenge.

Probiotics Not Antibiotics.

Crassostrea virginica (eastern oyster) is a species of oyster of significant economic importance in the Gulf of Mexico and Atlantic coasts of North America. In recent years stocks have been plagued by infections, especially in relation to juvenile and larval mortality. This leads to significant drops in recruitment, which can be crippling to the mariculture output. Amongst these pathogens are many Vibrio spp. and Roseovarius crassotstrea, the causative agent of juvenile oyster disease. One Vibrio of importance is Vibrio tubiashii, which causes virbiosis and attacks the larval stage. Good husbandry techniques such as regular water changes can help to avoid infection though this is still high risk for the fishery owners who favour the use of antibiotics. Antibiotics not only promote the evolution of resistance in pathogens but can also have damaging environmental effects in open system mariculture, for example they may affect symbiotic microbial species in wild populations.  One potential solution is the use of probiotics in place of antibiotics. These may of course have negative side effects for the environment and undoubtedly these will be uncovered, however the potential positives for aquaculture, coupled with the fact that a replacement for antibiotics must be found, make them an attractive alternative.

To test the effectiveness of some such probiotics, first potential probiotics need to be identified. The authors of this study did this through a series of bacteria-bacteria competition assays (colony-on-top assay & membrane overlay assay) with known pathogens and potential probiotics isolated from the natural habitat of the eastern oyster in Rhode Island. The most promising strains were Phaeobacter sp. S4 and Bacillus pumilus RI06-95 so these were brought forward to bacterial challenge trials involving juvenile and larval oysters (separately). Another aspect investigated was the longevity of the effects. This was done by the addition of pathogens at time intervals after removing the probiotics.

Both probiotic strains were found to significantly reduce mortality in bacterially challenged larvae and juveniles and neither had any known negative effects (mortality was tested for using controls). These benefits were achieved at a concentration that could realistically be achieved in commercial oyster cultures making the use of these probiotics feasible. The benefits, however, are not transferred from the probiotic to the organism long term so a constant supply of probiotics would be necessary. However with a short doubling time (roughly 3 hours in oyster culture medium) these may be self-sustaining depending on the flow through rate of the tank and available nutrients. One method of supply would be mixing probiotics with the feed organism, usually microalgae, though the first step towards this would be testing the effect of these strains on the feed organism(s). They also discovered that the initial screening process did not necessarily predict the in vivo effect as Bacillus pumilus did not inhibit the growth of V. tubiashi in vitro yet provided protection for both larvae and alga in vivo insinuating a symbiotic relationship or flaw in the assay process.

These findings further support the use of probiotics in aquaculture and could provide a more cost effective and environmentally friendly option over antibiotics. Keeping live cultures of probiotics on site could also help reduce the mounting costs of husbandry. Future studies should investigate what effects these probiotics have on feed organisms as well as the potential for use with other molluscan species that are affected by these pathogens.

Asymmetric division: a major cause of cyanobacteria cell death

Cyanobacteria have a significant role in global primary productivity. Forms Prochlorococcus and Synechococcus alone contribute up to 50% of total fixed carbon in low latitudes, a great ecological significance. An individual cell has four potential fates; division, eaten by predators, viral lysis or cell death. The cause of death will greatly influence the carbon flow within the food web. Cell death will lead to cell lysis and a release of dissolved organic carbon, as does viral lysis, but this is very difficult to measure. Many studies have classified cell death as programmed cell death (PCD) however this study promotes the idea of asymmetric division as the major cause.

Cyanobacteria colonies aim to optimize colony fitness therefore PCD would make evolutionary sense, where there is differentiation between cells, in order for different roles to be completed. However in some colonial forms, cell death and DNA fragmentation happens randomly, making PCD a less clear response. PCD is detected from increased caspase activity during cell death. It happens when the metabolism is forced beyond its limits, however, in Trichodesmium colonies approximately 30% of the colony survives during mass cell death. This proportion of the population is known as a “persister” fraction that is more resistant to environmental stress.

Asymmetric division occurs when daughter cells are morphologically or physiologically different which could lead to differing fitness. In Escherichia coli, one daughter cell has a reduced fitness where the parent cell has deposited all its metabolic waste, such as damaged proteins, resulting in an increased fitness and growth of the other daughter cell. Dead cells can also be regularly produced as a by-product and this reduces the burden of damaged proteins on the colony, thus improving overall fitness.

A large proportion of cyanobacteria occur in the top 50m of the water column where excess light can lead to cell death by protein damage. Cell death increases throughout the day and peaks in the late afternoon. Is it possible that the individuals which die in these situations have already accumulated metabolic damage from their parent cell and are therefore more susceptible to environmental conditions?

Protein damage can be discovered by measuring the protein carbonylation which needs to be tested in inclusion bodies produced asymmetrically by the parent cell and track their distribution. To conclude that asymmetric division is a major cause of cell death, determining the amount of oxidative damage along with the fate of a bacterial cell needs to be addressed. This knowledge will provide a better understanding of the whole food web structure and the carbon within it.

Franklin, D.J. (2014) Explaining the causes of cell death in cyanobacteria: what role for asymmetric division? Journal of Plankton Research. 36: 11-17

Bacterial heat shock proteins? Inducing invertebrate immune responses?!

   Previous posts discussing infection management in aquaculture have referred to the paradigm that invertebrates do not have acquired immune systems like us vertebrates. Without antibodies, how does the invertebrate immune system rely completely on innate (non-specific) defences? This study details an interesting mechanism of how the innate immune system responds to pathogens.

   Litopenaeus vannamei is a shrimp widely devoured by Homo sapiens and is assumed to have an innate immune system, defending the arthropod using melanisation, hemolymph clotting, foreign particle trapping and good old phagocytosis. The hemolymph enzyme transglutaminase (TGase) is instrumental in all of these processes and inhibiting its production in shrimp results in bacteria rich hemolymph and very sick crustaceans. Hemocytes produce prophenoloxidase (proPOase), the precursor to another important immune enzyme, phenoloxidase (POase). Once activated, PO facilitates phenol oxidation to antimicrobial quinones, which can also polymerize into melanin to encapsulate pathogens. Basically, TGase and proPO are what makes the crustacean innate immune system tick.

   As you know, heat shock proteins (HSPs) are a highly multifunctional family of proteins used by all organisms. The family HSP70 is the most highly conserved and studied; recently it has been flagged as a notable regulator in the initial innate immune system response, improving POase activity in Artemia infected with Vibrio. But such studies are controversial; many have used a recombinant HSP70 extracted from E. coli called DnaK, which may have been contaminated with endotoxins able to activate the immune system in a similar way.

   This study aimed to determine whether or not HSP70 homologue DnaK induces immune response in L. vannamei, by measuring changes in the expression of proPOase and TGase genes in response to an injection of either recombinant HSP70 (DnaK) from E. coli or a contaminant-free control of chemically synthesised HSP70 (synDnaK). All shrimp used were specific pathogen free (SPF) and given a high and a low dose injection. Gene expression was measured by synthesising cDNA from isolated RNA, then amplifying it by quantitative PCR with TGase and proPOase primers. TGase gene expression increased very significantly in response to low and high doses of DnaK, low doses of synDnaK, but did not change for high dose synDnaK. Expression of a proPOase gene increased significantly for low and high doses of DnaK; a similar pattern was observed for synDnaK. DnaK contamination was measured also, showing endotoxin levels much lower than concentrations found to induce crustacean immune response in other studies.

Recombinant DnaK and synthetic DnaK induced similar patterns of immune response, so contaminants are not the only contributors to stimulation of proPOase and TGase up-regulation. A novel mechanism for TGase and proPOase increase in response to pathogens has been demonstrated. Bizarrely, lower injections of both treatments induced stronger responses; this inverted dose-response relationship points to an unknown mechanism of immune system inhibition by DnaK. In conclusion, this study proposes the hypothesis that bacterial derived HSP70 acts as an alarm signal, activating host TGase and proPOase genes. I wonder how this response compares between organisms with only innate immune system and those with both acquired and innate immune system; is it stronger in the former because it is the main immune response? When pathogens initially colonise a host, they are probably exposed to a sudden range of stresses and therefore increase HSP production. This could be why the innate immune system has evolved to respond to HSP, because high bacterial HSP levels coincide with early infection.

Hu, B., Phuoc, L. H., Sorgeloos, P., & Bossier, P. (2014). Bacterial HSP70 (DnaK) is an efficient immune stimulator in Litopenaeus vannamei. Aquaculture, 418, 87-93.

Dormant V.cholerae cells are resuscitated by quorum sensing molecules

Detection of Vibrio cholerae in environmental samples is made difficult by its ability to enter a dormant ‘viable but nonculturable’ (VBNC) state. VBNC cells will persist in an environment but will not grow on traditional growth media, allowing them to go undetected by conventional techniques. Recently, fluorescent antibody based microscopy has allowed identification of V.cholerae in samples from which it would previous have gone unnoticed. Antibiotic selection techniques, using knowledge of the antibiotic resistance of previously cultured strains from pandemics, also allows small numbers of VBNC cells to be identified, by suppressing the growth of other bacteria.

The danger of VBNC cells is not just that they are difficult to detect, they can be ‘resuscitated’, switching from dormancy to an active state that is capable of infection. The researches had in a previous study identified two autoinducer molecules that were upregulated in conditions of large cells densities to promote the production of Vibrio extracellular polysaccharide by active cells. Because autoinducers are only produced under conditions of high cell density, they reasoned that they might also signal to dormant VBNC cells that conditions are favourable for growth, causing resuscitation.

They therefore proceeded to investigate whether biologically or synthetically produced autoinducer molecules would induce resuscitation of VBNC V.cholerae in environmental samples from Bangladesh.

Antibiotic selection techniques were used to identify samples that contained active V.cholerae, only samples that did not already contain active cells were used. They found that samples containing no culturable V.cholerae, when treated with spent media from either autoinducer-producing V.cholerae or E.coli, produced resuscitated cells within only a few hours of treatment. Water from the same samples tested negative for culturable V.cholerae following treatment with spent media from the controls: a V.cholerae strain that had had its autoinducer genes deleted and E.coli that contained the cloning vector used to induce autoinducer production but without the genes required for their production. Furthermore, profiling of the culturable V.cholerae strains produced via this resuscitation showed their close similarity to strains that had caused recent epidemics in the area.

Having established the link between autoinducers and resuscitation, they proceeded to investigate whether a wild type strain of V.cholerae could initiate resuscitation of VBNC cells. It could not without the addition of a recombinant plasmid containing synthase genes. Wild strains that already contain this plasmid do show resuscitation activity.

Chemically synthesized versions of the two autoinducer molecules caused resuscitation when provided together and individually, showing that the presence of only one of them is required for dormant cells to become active. One of the autoinducers is narrowly produced by Vibrios only, however the other is produced by many bacterial groups. This study therefore suggests that resuscitation of VBNC V.cholerae could occur either outside or inside the human host as a result of high concentrations of autoinducer molecules produced by environmental bacteria or those of the human microbiome. They suggest that this may explain the seasonality of cholera, as the seasonal occurrence of heterologous bacteria that produce the same autoinducer molecule may trigger resuscitation of large numbers of dormant V.cholerae. Another model would be that the bacteria of the human microbiome cause resuscitation upon ingestion, or that faecal bacteria that enter the environment are favoured by seasonal conditions and so induce resuscitation in the environment.

Many potential models are suggested, however, the major contribution of this paper is not its speculations but the mechanism it has identified that links resuscitation of dormant V.cholerae cells to bacterial inter-species communication via autoinducer molecules.

Bari, S. N., Roky, M. K., Mohiuddin, M., Kamruzzaman, M., Mekalanos, J. J., & Faruque, S. M. (2013). Quorum-sensing autoinducers resuscitate dormant Vibrio cholerae in environmental water samples. Proceedings of the National Academy of Sciences, 110(24), 9926-9931.

FARMED SHRIMPS FROM ASIA COULD BE CONTAMINATED WITH PATHOGENS OF VIBRIO SPP.

Popularity of shrimps as seafood has encouraged shrimp farming in South Asia and Latin America. We have been frequently coming across Vibrio spp. in recent lectures as very diverse group of bacteria, very dominant in the marine environment, associated with a number of diseases of corals, fish, crustaceans, marine mammals etc. and also of humans. As Colin explained in the last lecture, three Vibrio species Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus are of prime importance as they are causative agents of various disease outbreaks of humans. In intensive shrimp farming, antibiotics are heavily used, stimulating development of resistance among bacteria, including Vibrio spp. Similarly, antibiotics are also used to treat human diseases; for example, antibiotic treatment becomes necessity for patients of septicaemia caused by V. vulnificus. Here, aquaculture induced problem of antibiotic resistance, especially among human pathogens is notable and studies have shown resistant Vibrio spp. isolates from farmed shrimps.

Thailand has a huge aquaculture industry. Shrimps are farmed both in marine and inland waters. Producing shrimps also in inland waters with the use of antibiotics have had many ecological impacts, including impacts on bacteria like Vibrio spp. and their susceptibility to antibiotics. This study examined occurrence and antibiotic susceptibility of Vibrio pathogens of humans (V. cholerae, V. parahaemolyticus & V. vulnificus) in farmed shrimps from intensive farms of inland waters of Thailand.

A method called three-tube most-probable-number was used for isolating Vibrio species. Agar plating technique was used to culture the Vibrio pathogens and then colonies of bacteria were selected for biochemical tests, species-specific PCR and 16S rRNA sequencing. Total, 12 different antibiotics were tested in this study. Antibiotic resistant isolates were screened for resistant genes and those genes were sequenced using PCR.

The authors found that 94% of shrimp samples had V. cholerae. But, remarkably, none of them had cholera toxin. Thus, all the V. cholera isolated from farmed shrimps were non-choleragenic. Non-choleragenic strains of V. cholerae are abundant in estuarine environments. This study proposes that same is true for inland waters with shrimp aquaculture activity. Nevertheless, high frequencies and densities of V. cholera were found to be associated with the shrimps, capable of causing sporadic diarrhoea in humans.

V. parahaemolyticus was isolated from 38% of samples, of which one was positive for thermostable direct hemolysin (TDH). As Colin mentioned in the last lecture, TDH is produced by human disease producing strains of V. parahaemolyticus. This pathogen is found commonly in seafood and its contamination is a big problem in Asia. The authors also suggested a link between non-native species of shrimp (grown on farms in Thailand) which grows faster but found to have higher affinity for this pathogen.

The authors did not find notable concentrations of V. vulnificus from their samples. Nevertheless, they reported a previous study finding high concentration of V. vulnificus in farmed shrimps, sold in a market in China.

The authors discussed that shrimp aquaculture expansion in inland waters (and related lower salinity) might be a limiting factor for V. parahaemolyticus and V. vulnificus whereas this would be favourable for growth of V. cholerae. Nevertheless, shrimps grown in inland waters are at high risk of being contaminated with V. cholerae and V. parahaemolyticus. Other thing I want to mention here is that it is perhaps not very surprising to see Vibrios associated with shrimps because, as Colin have mentioned many times in the lectures that Vibrios prefer to colonise on chitinous organisms/chitin and have enzymes to breakdown chitin.

V. cholerae and V. parahaemolyticus mostly showed resistance to ampicillin and oxytetracyclin. V. vulnificus showed resistance to nalidixic acid. Farmed shrimps from inland waters can act as reservoirs of numerous oxytetracyclin-resistance genes, originating from both marine and inland waters. The resistance to oxytetracycline was related to plasmids which are passed from one bacteria to other in a process called horizontal gene transfer.

In summary, this study provides an experimental evidence for risk of contamination of Vibrio pathogens in farmed seafood. This paper attracted my attention because it links aquaculture, prevalence of bacteria (Vibrios) in these environments and seafood-borne bacterial diseases in humans. In this linkage, a worrisome factor is heavy use of antibiotics in aquaculture which is moving antibiotics in marine, freshwater and terrestrial environments and driving widespread antibiotic-resistance development among various bacteria (including many pathogens) from different environments to existing antibiotics.

Yano, Y., Hamano, K., Satomi, M., Tsutsui, I., Ban, M., & Aue-umneoy, D. (2014). Prevalence and antimicrobial susceptibility of Vibrio species related to food safety isolated from shrimp cultured at inland ponds in Thailand. Food Control, 38, 30-36.