Dual RNA-seq of zebrafish larvae infected with Shigella sonnei
ABSTRACT: Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries. Overall design: Profiling of host and pathogen transcriptome for zebrafish larvae infected with Shigella sonnei at 24 hours post infection and comparison to transcriptome of control injected larvae and control bacteria grown in liquid culture
Project description:Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.
Project description:Shigellosis (bacillary dysentery) is an acute enteric infection caused by members of Shigella genus. It causes annual deaths of approximately five million children in developing countries. Among Shigella spp., S. flexneri causes more serious forms of dysentery than other Shigella species. Due to the appearance of multidrug-resistant strains of Shigella spp., it is necessary to find alternative antimicrobial agents. The aims of this study were the isolation of a novel species-specific phage against S. flexneri and to evaluate its potential and efficacy for biocontrolling of S. flexneri in foods. Shigella flexneri PTCC 1234 was used as the host strain for bacteriophage isolation from waste water. A lytic phage of the Siphoviridae family was isolated and designated as vB_SflS-ISF001. The phage activity remained at high levels after 1 h of incubation at - 20 to 50 °C and was fairly stable for 1 h at pH values ranging from 7 to 9. The latent period and burst size were approximately 20 min and 53 ± 4 phages per host cell, respectively. Raw and cooked chicken breast were inoculated with a predetermined amount of S. flexneri and subjected to biocontrol test. The results showed that using vB_SflS-ISF001 phage led to more than two logs reduction in the count of viable S. flexneri. It was demonstrated that using vB_SflS-ISF001 phage is of high potential for developing an alternative strategy against S. flexneri contamination in foodstuffs.
Project description:Shigella flexneri is the primary cause of bacillary dysentery in the developing countries. S. flexneri serotype 1c is a novel serotype, which is found to be endemic in many developing countries, but little is known about its genomic architecture and virulence signatures. We have sequenced for the first time, the complete genome of S. flexneri serotype 1c strain Y394, to provide insights into its diversity and evolution.We generated a high-quality reference genome of S. flexneri serotype 1c using the hybrid methods of long-read single-molecule real-time (SMRT) sequencing technology and short-read MiSeq (Illumina) sequencing technology. The Y394 chromosome is 4.58 Mb in size and shares the basic genomic features with other S. flexneri complete genomes. However, it possesses unique and highly modified O-antigen structure comprising of three distinct O-antigen modifying gene clusters that potentially came from three different bacteriophages. It also possesses a large number of hypothetical unique genes compared to other S. flexneri genomes.Despite a high level of structural and functional similarities of Y394 genome with other S. flexneri genomes, there are marked differences in the pathogenic islands. The diversity in the pathogenic islands suggests that these bacterial pathogens are well adapted to respond to the selection pressures during their evolution, which might contribute to the differences in their virulence potential.
Project description:Shigella flexneri is the major pathogen causing bacillary dysentery in developing countries. S. flexneri is divided into at least 16 serotypes based on the combination of antigenic determinants present in the O-antigen. All the serotypes (except for serotype 6) share a basic O-unit containing one N-acetyl-d-glucosamine and three l-rhamnose residues, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation. Serotype Xv is a newly emerged and the most prevalent serotype in China, which can agglutinate with both MASF IV-1 and 7,8 monoclonal antibodies. The factor responsible for the presence of MASF IV-1 (E1037) epitope has not yet been identified. In this study, we analyzed the LPS structure of serotype Xv strains and found that the MASF IV-1 positive phenotype depends on an O-antigen modification with a phosphoethanolamine (PEtN) group attached at position 3 of one of the rhamnose residues. A plasmid carried gene, lpt-O (LPS phosphoethanolamine transferase for O-antigen), mediates the addition of PEtN for serotype Xv and other MASF IV-1 positive strains. These findings reveal a novel serotype conversion mechanism in S. flexneri and show the necessity of further extension of the serotype classification scheme recognizing the MASF IV-1 positive strains as distinctive subtypes.
Project description:The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery (blood in stool) worldwide every year, resulting in more than 200,000 deaths. A major challenge in combating bacillary dysentery is the lack of a small-animal model that recapitulates the symptoms observed in infected individuals, including bloody diarrhea. Here, we show that similar to humans, infant rabbits infected with S. flexneri experience severe inflammation, massive ulceration of the colonic mucosa, and bloody diarrhea. T3SS-dependent invasion of epithelial cells is necessary and sufficient for mediating immune cell infiltration and vascular lesions. However, massive ulceration of the colonic mucosa, bloody diarrhea, and dramatic weight loss are strictly contingent on the ability of the bacteria to spread from cell to cell. The infant rabbit model features bacterial dissemination as a critical determinant of S. flexneri pathogenesis and provides a unique small-animal model for research and development of therapeutic interventions.
Project description:Bacillary dysentery caused by Shigella flexneri is a major cause of under-five mortality in developing countries, where a novel S. flexneri serotype 1c has become very common since the 1980s. However, the origin and diversification of serotype 1c remain poorly understood. To understand the evolution of serotype 1c and their antimicrobial resistance, we sequenced and analyzed the whole-genome of 85 clinical isolates from the United Kingdom, Egypt, Bangladesh, Vietnam, and Japan belonging to serotype 1c and related serotypes of 1a, 1b and Y/Yv. We identified up to three distinct O-antigen modifying genes in S. flexneri 1c strains, which were acquired from three different bacteriophages. Our analysis shows that S. flexneri 1c strains have originated from serotype 1a and serotype 1b strains after the acquisition of bacteriophage-encoding gtrIc operon. The maximum-likelihood phylogenetic analysis using core genes suggests two distinct S. flexneri 1c lineages, one specific to Bangladesh, which originated from ancestral serotype 1a strains and the other from the United Kingdom, Egypt, and Vietnam originated from ancestral serotype 1b strains. We also identified 63 isolates containing multiple drug-resistant genes in them conferring resistance against streptomycin, sulfonamide, quinolone, trimethoprim, tetracycline, chloramphenicol, and beta-lactamase. Furthermore, antibiotic susceptibility assays showed 83 (97.6%) isolates as either complete or intermediate resistance to the WHO-recommended first- and second-line drugs. This changing drug resistance pattern demonstrates the urgent need for drug resistance surveillance and renewed treatment guidelines.
Project description:Shigella flexneri is a major cause of bacillary dysentery in Beijing, China. The genetic features and population structure of locally circulating clones remained unclear. In this study, we sequenced the genomes of 93 S. flexneri isolates from patients in Beijing from 2005 to 2018. Phylogenetic analysis revealed a predominant lineage comprised of ST100 isolates that had acquired an extensive repertoire of antimicrobial resistance determinants. A rapid local expansion of the largest clade of this lineage began in 2008 and gradually resulted in the dominance of serotype 2a. Other clades showed substantial evidence of interregional spread from other areas of China. Another lineage consisting of ST18 isolates was also identified and appeared to have persisted locally for nearly 6 decades. These findings suggest that S. flexneri epidemics in Beijing were caused by both local expansion and interregional transmission.IMPORTANCE Beijing is the largest transportation hub in China, with a highly mobile population. Shigella flexneri is a major cause of bacillary dysentery in Beijing. However, little is known about the genetic features and population structure of locally circulating S. flexneri clones. Whole-genome sequencing of 93 S. flexneri isolates revealed that S. flexneri epidemics in Beijing were predominantly caused by an ST100 clone. Interregional spread, rapid local expansion, and acquirement of antimicrobial resistance determinants have cocontributed to the epidemics of this clone. Another ST18 clone was also identified and showed long-term colonization in Beijing. Our study provides comprehensive insights into the population structure and evolutionary history of S. flexneri in Beijing.
Project description:Although the incidence of bacillary dysentery in China has been declining progressively, a considerable disease burden still exists. Few studies have analyzed bacillary dysentery across China and knowledge gaps still exist in the aspects of geographic distribution and ecological drivers, seasonality and its association with meteorological factors, urban-rural disparity, prevalence and distribution of Shigella species. Here, we performed nationwide analyses to fill the above gaps. Geographically, we found that incidence increased along an east-west gradient which was inversely related to the economic conditions of China. Two large endemically high-risk regions in western China and their ecological drivers were identified for the first time. We characterized seasonality of bacillary dysentery incidence and assessed its association with meteorological factors, and saw that it exhibits north-south differences in peak duration, relative amplitude and key meteorological factors. Urban and rural incidences among China's cities were compared, and disparity associated with urbanization level was invariant in most cities. Balanced decrease of urban and rural incidence was observed for all provinces except Hunan. S. flexneri and S. sonnei were identified as major causative species. Increasing prevalence of S. sonnei and geographic distribution of Shigella species were associated with economic status. Findings and inferences from this study draw broader pictures of bacillary dysentery in mainland China and could provide useful information for better interventions and public health planning.
Project description:Shigella infections account for a considerable burden of acute diarrheal diseases worldwide and remain a major cause of childhood mortality in developing countries. Although, all four species of Shigella (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) cause bacillary dysentery, historically only S. dysenteriae type 1 has been recognized as carrying the genes for Shiga toxin (stx). Recent epidemiological data, however, have suggested that the emergence of stx carrying S. flexneri strains may have originated from bacteriophage-mediated inter-species horizontal gene transfer in one specific geographical area, Hispaniola. To test this hypothesis, we analyzed whole genome sequences of stx-encoding phages carried by S. flexneri strains isolated in Haiti and S. flexneri S. boydii and S. dysenteriae strains isolated from international travelers who likely acquired the infection in Haiti or the Dominican Republic. Phylogenetic analysis showed that phage sequences encoded in the Shigella strains from Hispaniola were bacteriophage ?POC-J13 and they were all closely related to a phage isolated from a USA isolate, E. coli 2009C-3133 serotype O119:H4. In addition, despite the low genetic heterogeneity of phages from different Shigella spp. circulating in the Caribbean island between 2001 and 2014, two distinct clusters emerged in Haiti and the Dominican Republic. Each cluster possibly originated from phages isolated from S. flexneri 2a, and within each cluster several instances of horizontal phage transfer from S. flexneri 2a to other species were detected. The implications of the emergence of stx-producing non-S. dysenteriae type 1 Shigella species, such as S. flexneri, spans not only the basic science behind horizontal phage spread, but also extends to medical treatment of patients infected with this pathogen.
Project description:Shigella flexneri, the etiological agent of bacillary dysentery, invades the human colonic epithelium and causes its massive inflammatory destruction. Little is known about the post-translational modifications implicated in regulating the host defense pathway against Shigella. Here, we show that SUMO-2 impairs Shigella invasion of epithelial cells in vitro. Using mice haploinsufficient for the SUMO E2 enzyme, we found that sumoylation regulates intestinal permeability and is required to restrict epithelial invasion and control mucosal inflammation. Quantitative proteomics reveals that Shigella infection alters the sumoylation status of a restricted set of transcriptional regulators involved in intestinal functions and inflammation. Consistent with this, sumoylation restricts the pro-inflammatory transcriptional response of Shigella-infected guts. Altogether, our results show that the SUMO pathway is an essential component of host innate protection, as it reduces the efficiency of two key steps of shigellosis: invasion and inflammatory destruction of the intestinal epithelium.