The Impact of Protozoan Predation on the Pathogenicity of Vibrio cholerae

© Copyright © 2020 Espinoza-Vergara, Hoque, McDougald and Noorian. 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

Environmental prevalence of toxigenic Vibrio cholerae O1 in Bangladesh coincides with V. cholerae non-O1 non-O139 genetic variants which overproduce autoinducer-2.

Prevalence of toxigenic Vibrio cholerae O1 in aquatic reservoirs in Bangladesh apparently increases coinciding with the occurrence of seasonal cholera epidemics. In between epidemics, these bacteria persist in water mostly as dormant cells, known as viable but non-culturable cells (VBNC), or conditionally viable environmental cells (CVEC), that fail to grow in routine culture. CVEC resuscitate to active cells when enriched in culture medium supplemented with quorum sensing autoinducers CAI-1 or AI-2 which are signal molecules that regulate gene expression dependent on cell density. V. cholerae O1 mutant strains with inactivated cqsS gene encoding the CAI-1 receptor has been shown to overproduce AI-2 that enhance CVEC resuscitation in water samples. Since V. cholerae non-O1 non-O139 (non-cholera-vibrios) are abundant in aquatic ecosystems, we identified and characterized naturally occurring variant strains of V. cholerae non-O1 non-O139 which overproduce AI-2, and monitored their co-occurrence with V. cholerae O1 in water samples. The nucleotide sequence and predicted protein products of the cqsS gene carried by AI-2 overproducing variant strains showed divergence from that of typical V. cholerae O1 or non-O1 strains, and their culture supernatants enhanced resuscitation of CVEC in water samples. Furthermore, prevalence of V. cholerae O1 in the aquatic environment was found to coincide with an increase in AI-2 overproducing non-O1 non-O139 strains. These results suggest a possible role of non-cholera vibrios in the environmental biology of the cholera pathogen, in which non-O1 non-O139 variant strains overproducing AI-2 presumably contribute in resuscitation of the latent pathogen, leading to seasonal cholera epidemics. Importance. Toxigenic Vibrio cholerae which causes seasonal epidemics of cholera persists in aquatic reservoirs in endemic areas. The bacteria mostly exist in a dormant state during inter-epidemic periods, but periodically resuscitate to the active form. The resuscitation is enhanced by signal molecules called autoinducers (AIs). Toxigenic V. cholerae can be recovered from water samples that normally test negative for the organism in conventional culture, by supplementing the culture medium with exogenous AIs. V. cholerae belonging to the non-O1 non-O139 serogroups which do not cause cholera are also abundant in natural waters, and they are capable of producing AIs. In this study we characterized V. cholerae non-O1 non-O139 variant strains which overproduce an autoinducer called AI-2, and found that the abundance of the cholera pathogen in aquatic reservoirs correlates with an increase in the AI-2 overproducing strains. Our results suggest a probable role of these variant strains in the environmental biology and epidemiology of toxigenic V. cholerae, and may lead to novel means for surveillance, prevention and control of cholera

Analysis of the CRISPR-Cas system in bacteriophages active on epidemic strains of Vibrio cholerae in Bangladesh.

CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated proteins) are microbial nuclease systems involved in defense against phages. Bacteria also resist phages by hosting phage-inducible chromosomal islands (PICI) which prevent phage reproduction. Vibrio cholerae which causes cholera epidemics, interacts with numerous phages in the environment and in cholera patients. Although CRISPR-Cas systems are usually carried by bacteria and archea, recently V. cholerae specific ICP1 phages were found to host a CRISPR-Cas system that inactivates PICI-like elements (PLE) in V. cholerae. We analyzed a collection of phages and V. cholerae isolated during seasonal cholera epidemics in Bangladesh, to study the distribution, and recent evolution of the phage-encoded CRISPR-Cas system. Five distinct but related phages carrying the CRISPR-Cas system, and possible CRISPR-Cas negative progenitor phages were identified. Furthermore, CRISPR arrays in the phages were found to have evolved by acquisition of new spacers targeting diverse regions of PLEs carried by the V. cholerae strains, enabling the phages to efficiently grow on PLE positive strains. Our results demonstrate a continuing arms-race involving genetic determinants of phage-resistance in V. cholerae, and the phage-encoded CRISPR-Cas system in the co-evolution of V. cholerae and its phages, presumably fostered by their enhanced interactions during seasonal epidemics of cholera

Environmental bacteriophages active on biofilms and planktonic forms of toxigenic Vibrio cholerae: Potential relevance in cholera epidemiology.

METHODS: Phages isolated from environmental waters in Bangladesh were tested for their host specificity towards V. cholerae O1 and O139, and the ability to disperse V. cholerae biofilms formed in the laboratory. Representative phages were further characterized by electron microscopy and whole genome sequencing. Selected phages were then introduced in various combinations to biofilms of toxigenic V. cholerae added to samples of river water, and the dispersion of biofilms as well as the growth kinetics of V. cholerae and the phages were monitored. RESULTS: A phage cocktail composed of three different phages isolated from surface waters in Bangladesh and designated as JSF7, JSF4, and JSF3 could significantly influence the distribution and concentration of the active planktonic form and biofilm associated form of toxigenic V. cholerae in water. While JSF7 showed a biofilm degrading activity and dispersed cells from both V. cholerae O1 and O139 derived biofilms thus increasing the concentration of planktonic V. cholerae in water, JSF4 and JSF3 showed strong bactericidal activity against V. cholerae O1 and O139 respectively. A mixture of all three phages could effectively reduce both biofilm-associated and planktonic V. cholerae in river water microcosms. SIGNIFICANCE: Besides potential applicability in phage-mediated control of cholera, our results have relevance in appreciating possible intricate role of diverse environmental phages in the epidemiology of the disease, since both biofilms and phages influence the prevalence and infectivity of V. cholerae in a variety of ways

Quorum Regulated Resistance of Vibrio cholerae against Environmental Bacteriophages.

Predation by bacteriophages can significantly influence the population structure of bacterial communities. Vibrio cholerae the causative agent of cholera epidemics interacts with numerous phages in the aquatic ecosystem, and in the intestine of cholera patients. Seasonal epidemics of cholera reportedly collapse due to predation of the pathogen by phages. However, it is not clear how sufficient number of the bacteria survive to seed the environment in the subsequent epidemic season. We found that bacterial cell density-dependent gene expression termed "quorum sensing" which is regulated by signal molecules called autoinducers (AIs) can protect V. cholerae against predatory phages. V. cholerae mutant strains carrying inactivated AI synthase genes were significantly more susceptible to multiple phages compared to the parent bacteria. Likewise when mixed cultures of phage and bacteria were supplemented with exogenous autoinducers CAI-1 or AI-2 produced by recombinant strains carrying cloned AI synthase genes, increased survival of V. cholerae and a decrease in phage titer was observed. Mutational analyses suggested that the observed effects of autoinducers are mediated in part through the quorum sensing-dependent production of haemaglutinin protease, and partly through downregulation of phage receptors. These results have implication in developing strategies for phage mediated control of cholera

The Effect of Arsenic Mitigation Interventions on Disease Burden in Bangladesh

Many interventions have been advocated to mitigate the impact of arsenic contamination of drinking water in Bangladesh. However, there are few data on the true magnitude of arsenic-related disease in Bangladesh nationally. There has also been little consideration given to possible adverse effects of such interventions, in particular, diarrheal disease. The purpose of this study was to estimate and compare the likely impacts of arsenic mitigation interventions on both arsenic-related disease and water-borne infectious disease. We found that arsenic-related disease currently results in 9,136 deaths per year and 174,174 disability-adjusted life years (DALYs; undiscounted) lost per year in those exposed to arsenic concentrations > 50 μg/L. This constitutes 0.3% of the total disease burden in Bangladesh in terms of undiscounted DALYs. We found intervention to be of overall benefit in reducing disease burden in most scenarios examined, but the concomitant increase in water-related infectious disease significantly reduced the potential benefits gained from intervention. A minimum reduction in arsenic-related DALYs of 77% was necessary before intervention achieved any reduction in net disease burden. This is assuming that interventions were provided to those exposed to > 50 μg/L and would concomitantly result in a 20% increase in water-related infectious disease in those without access to adequate sanitation. Intervention appears to be justified for those populations exposed to high levels of arsenic, but it must be based on exposure levels and on the effectiveness of interventions not only in reducing arsenic but in minimizing risk of water-related infections

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

Zeolite synthesis from low-cost materials and environmental applications: A review

Zeolites with the three-dimensional structures occur naturally or can be synthesized in the laboratory. Zeolites have versatile applications such as environmental remediation, catalytic activity, biotechnological application, gas sensing and medicinal applications. Although, naturally occurring zeolites are readily available, nowadays, more emphasis is given on the synthesis of the zeolites due to their easy synthesis in the pure form, better ion exchange capabilities and uniform in size. Recently, much attention has also been paid on how zeolite is being synthesized from low-cost material (e.g., rice husk), particularly, by resolving the major environmental issues. Hence, the main purpose of this review is to make an effective resolution of zeolite synthesis methods together with potential applications in environmental engineering. Among different synthesis methods, hydrothermal method is commonly found to be used widely in the synthesis of various zeolites from inexpensive raw materials such as fly ash, rice husk ash, blast furnace slag, municipal solid waste, paper sludge, lithium slag and kaolin. Besides, future expectation in the field of synthetic zeolites research is also included

Theoretical study of Oldroyd-b visco-elastic fluid flow through curved pipes with slip effects in polymer flow processing

The characteristics of the flow field of both viscous and viscoelastic fluids passing through a curved pipe with a Navier slip boundary condition have been investigated analytically in the present study. The Oldroyd-B constitutive equation is employed to simulate realistic transport of dilute polymeric solutions in curved channels. In order to linearize the momentum and constitutive equations, a perturbation method is used in which the ratio of radius of cross section to the radius of channel curvature is employed as the perturbation parameter. The intensity of secondary and main flows is mainly affected by the hoop stress and it is demonstrated in the present study that both the Weissenberg number (the ratio of elastic force to viscous force) and slip coefficient play major roles in determining the strengths of both flows. It is also shown that as a result of an increment in slip coefficient, the position of maximum velocity markedly migrates away from the pipe center towards the outer side of curvature. Furthermore, results corresponding to Navier slip scenarios exhibit non-uniform distributions in both the main and lateral components of velocity near the wall which can notably vary from the inner side of curvature to the outer side. The present solution is also important in polymeric flow processing systems because of experimental evidence indicating that the no-slip condition can fail for these flows, which is of relevance to chemical engineers

Genome wide in silico SNP-tumor association analysis

BACKGROUND: Carcinogenesis occurs, at least in part, due to the accumulation of mutations in critical genes that control the mechanisms of cell proliferation, differentiation and death. Publicly accessible databases contain millions of expressed sequence tag (EST) and single nucleotide polymorphism (SNP) records, which have the potential to assist in the identification of SNPs overrepresented in tumor tissue. METHODS: An in silico SNP-tumor association study was performed utilizing tissue library and SNP information available in NCBI's dbEST (release 092002) and dbSNP (build 106). RESULTS: A total of 4865 SNPs were identified which were present at higher allele frequencies in tumor compared to normal tissues. A subset of 327 (6.7%) SNPs induce amino acid changes to the protein coding sequences. This approach identified several SNPs which have been previously associated with carcinogenesis, as well as a number of SNPs that now warrant further investigation CONCLUSIONS: This novel in silico approach can assist in prioritization of genes and SNPs in the effort to elucidate the genetic mechanisms underlying the development of cancer