Increased iron utilization and oxidative stress tolerance in a Vibrio cholerae flrA mutant confers resistance to amoeba predation.

IMPORTANCE: Persistence of V. cholerae in the aquatic environment contributes to the fatal diarrheal disease cholera, which remains a global health burden. In the environment, bacteria face predation pressure by heterotrophic protists such as the free-living amoeba A. castellanii. This study explores how a mutant of V. cholerae adapts to acquire essential nutrients and survive predation. Here, we observed that up-regulation of iron acquisition genes and genes regulating resistance to oxidative stress enhances pathogen fitness. Our data show that V. cholerae can defend predation to overcome nutrient limitation and oxidative stress, resulting in an enhanced survival inside the protozoan hosts.

Chemically Mediated Interactions with Macroalgae Negatively Affect Coral Health but Induce Limited Changes in Coral Microbiomes.

Allelopathic chemicals facilitated by the direct contact of macroalgae with corals are potentially an important mechanism mediating coral-macroalgal interactions, but only a few studies have explored their impacts on coral health and microbiomes and the coral's ability to recover. We conducted a field experiment on an equatorial urbanized reef to assess the allelopathic effects of four macroalgal species (Bryopsis sp., Endosiphonia horrida, Hypnea pannosa and Lobophora challengeriae) on the health and microbiomes of three coral species (Merulina ampliata, Montipora stellata and Pocillopora acuta). Following 24 h of exposure, crude extracts of all four macroalgal species caused significant coral tissue bleaching and reduction in effective quantum yield. The corals were able to recover within 72 h of the removal of extracts, except those that were exposed to L. challengeriae. While some macroalgal extracts caused an increase in the alpha diversity of coral microbiomes, there were no significant differences in the composition and variability of coral microbiomes between controls and macroalgal extracts at each sampling time point. Nevertheless, DESeq2 differential abundance analyses showed species-specific responses of coral microbiomes. Overall, our findings provide insights on the limited effect of chemically mediated interactions with macroalgae on coral microbiomes and the capacity of corals to recover quickly from the macroalgal chemicals.

Protozoan predation as a driver of diversity and virulence in bacterial biofilms.

Protozoa are eukaryotic organisms that play a crucial role in nutrient cycling and maintaining balance in the food web. Predation, symbiosis and parasitism are three types of interactions between protozoa and bacteria. However, not all bacterial species are equally susceptible to protozoan predation as many are capable of defending against predation in numerous ways and may even establish either a symbiotic or parasitic life-style. Biofilm formation is one such mechanism by which bacteria can survive predation. Structural and chemical components of biofilms enhance resistance to predation compared to their planktonic counterparts. Predation on biofilms gives rise to phenotypic and genetic heterogeneity in prey that leads to trade-offs in virulence in other eukaryotes. Recent advances, using molecular and genomics techniques, allow us to generate new information about the interactions of protozoa and biofilms of prey bacteria. This review presents the current state of the field on impacts of protozoan predation on biofilms. We provide an overview of newly gathered insights into (i) molecular mechanisms of predation resistance in biofilms, (ii) phenotypic and genetic diversification of prey bacteria, and (iii) evolution of virulence as a consequence of protozoan predation on biofilms.

Environmental Reservoirs of Pathogenic Vibrio spp. and Their Role in Disease: The List Keeps Expanding.

Vibrio species are natural inhabitants of aquatic environments and have complex interactions with the environment that drive the evolution of traits contributing to their survival. These traits may also contribute to their ability to invade or colonize animal and human hosts. In this review, we attempt to summarize the relationships of Vibrio spp. with other organisms in the aquatic environment and discuss how these interactions could potentially impact colonization of animal and human hosts.

Associational Resistance to Predation by Protists in a Mixed Species Biofilm.

Mixed species biofilms exhibit increased tolerance to numerous stresses compared to single species biofilms. The aim of this study was to examine the effect of grazing by the heterotrophic protist, Tetrahymena pyriformis, on a mixed species biofilm consisting of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Protozoan grazing significantly reduced the single species K. pneumoniae biofilm, and the single species P. protegens biofilm was also sensitive to grazing. In contrast, P. aeruginosa biofilms were resistant to predation. This resistance protected the otherwise sensitive members of the mixed species biofilm consortium. Rhamnolipids produced by P. aeruginosa were shown to be the primary toxic factor for T. pyriformis. However, a rhamnolipid-deficient mutant of P. aeruginosa (P. aeruginosa ΔrhlAB) maintained grazing resistance in the biofilm, suggesting the presence of at least one additional protective mechanism. P. aeruginosa with a deleted gene encoding the type III secretion system also resisted grazing. A transposon library was generated in the ΔrhlAB mutant to identify the additional factor involved in community biofilm protection. Results indicated that the Pseudomonas Quinolone Signal (PQS), a quorum sensing signaling molecule, was likely responsible for this effect. We confirmed this observation by showing that double mutants of ΔrhlAB and genes in the PQS biosynthetic operon lost grazing protection. We also showed that PQS was directly toxic to T. pyriformis. This study demonstrates that residing in a mixed species biofilm can be an advantageous strategy for grazing sensitive bacterial species, as P. aeruginosa confers community protection from protozoan grazing through multiple mechanisms. IMPORTANCE Biofilms have been shown to protect bacterial cells from predation by protists. Biofilm studies have traditionally used single species systems, which have provided information on the mechanisms and regulation of biofilm formation and dispersal, and the effects of predation on these biofilms. However, biofilms in nature are comprised of multiple species. To better understand how multispecies biofilms are impacted by predation, a model mixed-species biofilm was here exposed to protozoan predation. We show that the grazing sensitive strains K. pneumonia and P. protogens gained associational resistance from the grazing resistant P. aeruginosa. Resistance was due to the secretion of rhamnolipids and quorum sensing molecule PQS. This work highlights the importance of using mixed species systems.

Bacterial biofilm colonization and succession in tropical marine waters are similar across different types of stone materials used in seawall construction.

Seawalls are important in protecting coastlines from currents, erosion, sea-level rise, and flooding. They are, however, associated with reduced biodiversity, due to their steep orientation, lack of microhabitats, and the materials used in their construction. Hence, there is considerable interest in modifying seawalls to enhance the settlement and diversity of marine organisms, as microbial biofilms play a critical role facilitating algal and invertebrate colonization. We assessed how different stone materials, ranging from aluminosilicates to limestone and concrete, affect biofilm formation. Metagenomic assessment of marine microbial communities indicated no significant impact of material on microbial diversity, irrespective of the diverse surface chemistry and topography. Based on KEGG pathway analysis, surface properties appeared to influence the community composition and function during the initial stages of biofilm development, but this effect disappeared by Day 31. We conclude that marine biofilms converged over time to a generic marine biofilm, rather than the underlying stone substrata type playing a significant role in driving community composition.

Protozoal food vacuoles enhance transformation in Vibrio cholerae through SOS-regulated DNA integration.

Vibrio cholerae, the bacterial pathogen responsible for the diarrheal disease cholera, resides in the aquatic environment between outbreaks. For bacteria, genetic variation by lateral gene transfer (LGT) is important for survival and adaptation. In the aquatic environment, V. cholerae is predominantly found in biofilms associated with chitinous organisms or with chitin "rain". Chitin induces competency in V. cholerae, which can lead to LGT. In the environment, V. cholerae is also subjected to predation pressure by protist. Here we investigated whether protozoal predation affected LGT using the integron as a model. Integrons facilitate the integration of mobile DNA (gene cassettes) into the bacterial chromosome. We report that protozoal predation enhances transformation of a gene cassette by as much as 405-fold. We show that oxidative radicals produced in the protozoal phagosome induces the universal SOS response, which in turn upregulates the integron-integrase, the recombinase that facilitates cassette integration. Additionally, we show that during predation, V. cholerae requires the type VI secretion system to acquire the gene cassette from Escherichia coli. These results show that protozoal predation enhances LGT thus producing genetic variants that may have increased capacity to survive grazing. Additionally, the conditions in the food vacuole may make it a "hot spot" for LGT by accumulating diverse bacteria and inducing the SOS response helping drive genetic diversification and evolution.

Adaptation to an Amoeba Host Leads to Pseudomonas aeruginosa Isolates with Attenuated Virulence.

The opportunistic pathogen Pseudomonas aeruginosa is ubiquitous in the environment, and in humans, it is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when coincubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence for the nematode Caenorhabditis elegans was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and nonadapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoebae as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoebae resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water, where bacteria are constantly under threat of being consumed by bacterial predators, e.g., protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examined the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa. We show that long-term coincubation with protozoa gave rise to mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to those observed in isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types as well as similar adaptation strategies.

Loss of the Acetate Switch in Vibrio vulnificus Enhances Predation Defense against Tetrahymena pyriformis.

Vibrio vulnificus is an opportunistic human pathogen and autochthonous inhabitant of coastal marine environments, where the bacterium is under constant predation by heterotrophic protists or protozoans. As a result of this selection pressure, genetic variants with antipredation mechanisms are selected for and persist in the environment. Such natural variants may also be pathogenic to animal or human hosts, making it important to understand these defense mechanisms. To identify antipredator strategies, 13 V. vulnificus strains of different genotypes isolated from diverse environments were exposed to predation by the ciliated protozoan Tetrahymena pyriformis, and only strain ENV1 was resistant to predation. Further investigation of the cell-free supernatant showed that ENV1 acidifies the environment by the excretion of organic acids, which are toxic to T. pyriformis. As this predation resistance was dependent on the availability of iron, transcriptomes of V. vulnificus in iron-replete and iron-deplete conditions were compared. This analysis revealed that ENV1 ferments pyruvate and the resultant acetyl-CoA leads to acetate synthesis under aerobic conditions, a hallmark of overflow metabolism. The anaerobic respiration global regulator arcA was upregulated when iron was available. An ΔarcA deletion mutant of ENV1 accumulated less acetate and, importantly, was sensitive to grazing by T. pyriformis. Based on the transcriptome response and quantification of metabolites, we conclude that ENV1 has adapted to overflow metabolism and has lost a control switch that shifts metabolism from acetate excretion to acetate assimilation, enabling it to excrete acetate continuously. We show that overflow metabolism and the acetate switch contribute to prey-predator interactions. IMPORTANCE Bacteria in the environment, including Vibrio spp., interact with protozoan predators. To defend against predation, bacteria evolve antipredator mechanisms ranging from changing morphology, biofilm formation, and secretion of toxins or virulence factors. Some of these adaptations may result in strains that are pathogenic to humans. Therefore, it is important to study predator defense strategies of environmental bacteria. V. vulnificus thrives in coastal waters and infects humans. Very little is known about the defense mechanisms V. vulnificus expresses against predation. Here, we show that a V. vulnificus strain (ENV1) has rewired the central carbon metabolism, enabling the production of excess organic acid that is toxic to the protozoan predator T. pyriformis. This is a previously unknown mechanism of predation defense that protects against protozoan predators.

Adaptation to an amoeba host drives selection of virulence-associated traits in Vibrio cholerae.

Predation by heterotrophic protists drives the emergence of adaptive traits in bacteria, and often these traits lead to altered interactions with hosts and persistence in the environment. Here we studied adaptation of the cholera pathogen, Vibrio cholerae during long-term co-incubation with the protist host, Acanthamoeba castellanii. We determined phenotypic and genotypic changes associated with long-term intra-amoebal host adaptation and how this impacts pathogen survival and fitness. We showed that adaptation to the amoeba host leads to temporal changes in multiple phenotypic traits in V. cholerae that facilitate increased survival and competitive fitness in amoeba. Genome sequencing and mutational analysis revealed that these altered lifestyles were linked to non-synonymous mutations in conserved regions of the flagellar transcriptional regulator, flrA. Additionally, the mutations resulted in enhanced colonisation in zebrafish, establishing a link between adaptation of V. cholerae to amoeba predation and enhanced environmental persistence. Our results show that pressure imposed by amoeba on V. cholerae selects for flrA mutations that serves as a key driver for adaptation. Importantly, this study provides evidence that adaptive traits that evolve in pathogens in response to environmental predatory pressure impact the colonisation of eukaryotic organisms by these pathogens.

Differential Response of the Microbiome of Pocillopora acuta to Reciprocal Transplantation Within Singapore.

As corals continue to decline globally, particularly due to climate change, it is vital to understand the extent to which their microbiome may confer an adaptive resilience against environmental stress. Corals that survive on the urban reefs of Singapore are ideal candidates to study the association of scleractinians with their microbiome, which in turn can inform reef conservation and management. In this study, we monitored differences in the microbiome of Pocillopora acuta colonies reciprocally transplanted between two reefs, Raffles and Kusu, within the Port of Singapore, where corals face intense anthropogenic impacts. Pocillopora acuta had previously been shown to host distinct microbial communities between these two reefs. Amplicon sequencing (16S rRNA) was used to assess the coral microbiomes at 1, 2, 4, and 10 days post-transplantation. Coral microbiomes responded rapidly to transplantation, becoming similar to those of the local corals at the destination reef within one day at Raffles and within two days at Kusu. Elevated nitrate concentrations were detected at Raffles for the duration of the study, potentially influencing the microbiome's response to transplantation. The persistence of corals within the port of Singapore highlights the ability of corals to adapt to stressful environments. Further, coral resilience appears to coincide with a dynamic microbiome which can undergo shifts in composition without succumbing to dysbiosis.

Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants.

BACKGROUND: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. RESULTS: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. CONCLUSIONS: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation.

The Repressor C Protein, Pf4r, Controls Superinfection of Pseudomonas aeruginosa PAO1 by the Pf4 Filamentous Phage and Regulates Host Gene Expression.

It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in Pseudomonas aeruginosa PAO1. These behaviours are linked to the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homologue of the P2 phage repressor C and was recently named Pf4r. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show a critical site in repressor C (Pf4r) where a mutation in the site, 788799A>G (Ser4Pro), causes Pf4r to lose its function as the immunity factor against reinfection by Pf4. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the E.coli bacteriophage P2 RepC protein. A mutation, Pf4r*, associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by multi-angle light scattering (MALS) analysis, where the Pf4r* protein only forms monomers. The loss of dimerisation also explains the loss of Pf4r's immunity function. Phenotypic assays showed that Pf4r increased LasB activity and was also associated with a slight increase in the percentage of morphotypic variants. ChIPseq and transcriptomic analyses suggest that Pf4r also likely functions as a transcriptional regulator for other host genes. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in P. aeruginosa biofilm development.

Microbial predation accelerates granulation and modulates microbial community composition.

BACKGROUND: Bacterial communities are responsible for biological nutrient removal and flocculation in engineered systems such as activated floccular sludge. Predators such as bacteriophage and protozoa exert significant predation pressure and cause bacterial mortality within these communities. However, the roles of bacteriophage and protozoan predation in impacting granulation process remain limited. Recent studies hypothesised that protozoa, particularly sessile ciliates, could have an important role in granulation as these ciliates were often observed in high abundance on surfaces of granules. Bacteriophages were hypothesized to contribute to granular stability through bacteriophage-mediated extracellular DNA release by lysing bacterial cells. This current study investigated the bacteriophage and protozoan communities throughout the granulation process. In addition, the importance of protozoan predation during granulation was also determined through chemical killing of protozoa in the floccular sludge. RESULTS: Four independent bioreactors seeded with activated floccular sludge were operated for aerobic granulation for 11 weeks. Changes in the phage, protozoa and bacterial communities were characterized throughout the granulation process. The filamentous phage, Inoviridae, increased in abundance at the initiation phase of granulation. However, the abundance shifted towards lytic phages during the maturation phase. In contrast, the abundance and diversity of protozoa decreased initially, possibly due to the reduction in settling time and subsequent washout. Upon the formation of granules, ciliated protozoa from the class Oligohymenophorea were the dominant group of protozoa based on metacommunity analysis. These protozoa had a strong, positive-correlation with the initial formation of compact aggregates prior to granule development. Furthermore, chemical inhibition of these ciliates in the floccular sludge delayed the initiation of granule formation. Analysis of the bacterial communities in the thiram treated sludge demonstrated that the recovery of 'Candidatus Accumulibacter' was positively correlated with the formation of compact aggregates and granules. CONCLUSION: Predation by bacteriophage and protozoa were positively correlated with the formation of aerobic granules. Increases in Inoviridae abundance suggested that filamentous phages may promote the structural formation of granules. Initiation of granules formation was delayed due to an absence of protozoa after chemical treatment. The presence of 'Candidatus Accumulibacter' was necessary for the formation of granules in the absence of protozoa.

The Impact of Protozoan Predation on the Pathogenicity of Vibrio cholerae.

In the aquatic environment, Vibrio spp. interact with many living organisms that can serve as a replication niche, including heterotrophic protists, or protozoa. Protozoa engulf bacteria and package them into phagosomes where the cells are exposed to low pH, antimicrobial peptides, reactive oxygen/nitrogen species, proteolytic enzymes, and low concentrations of essential metal ions such as iron. However, some bacteria can resist these digestive processes. For example, Vibrio cholerae and Vibrio harveyi can resist intracellular digestion. In order to survive intracellularly, bacteria have acquired and/or developed specific factors that help them to resist the unfavorable conditions encountered inside of the phagosomes. Many of these intra-phagosomal factors used to kill and digest bacteria are highly conserved between eukaryotic cells and thus are also expressed by the innate immune system in the gastrointestinal tract as the first line of defense against bacterial pathogens. Since pathogenic bacteria have been shown to be hypervirulent after they have passed through protozoa, the resistance to digestion by protist hosts in their natural environment plays a key role in enhancing the infectious potential of pathogenic Vibrio spp. This review will investigate the current knowledge in interactions of bacteria with protozoa and human host to better understand the mechanisms used by both protozoa and human hosts to kill bacteria and the bacterial response to them.

Vibrio cholerae residing in food vacuoles expelled by protozoa are more infectious in vivo.

Vibrio cholerae interacts with many organisms in the environment, including heterotrophic protists (protozoa). Several species of protozoa have been reported to release undigested bacteria in expelled food vacuoles (EFVs) when feeding on some pathogens. While the production of EFVs has been reported, their biological role as a vector for the transmission of pathogens remains unknown. Here we report that ciliated protozoa release EFVs containing V. cholerae. The EFVs are stable, the cells inside them are protected from multiple stresses, and large numbers of cells escape when incubated at 37 °C or in the presence of nutrients. We show that OmpU, a major outer membrane protein positively regulated by ToxR, has a role in the production of EFVs. Notably, cells released from EFVs have growth and colonization advantages over planktonic cells both in vitro and in vivo. Our results suggest that EFVs facilitate V. cholerae survival in the environment, enhancing their infectious potential and may contribute to the dissemination of epidemic V. cholerae strains. These results improve our understanding of the mechanisms of persistence and the modes of transmission of V. cholerae and may further apply to other opportunistic pathogens that have been shown to be released by protists in EFVs.