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The emergence and dissemination of antibiotic resistance genes (ARGs) in the ecosystem are global public health concerns. One Health emphasizes the interconnectivity between different habitats and seeks to optimize animal, human, and environmental health. However, information on the dissemination of antibiotic resistance genes (ARGs) within complex microbiomes in natural habitats is scarce. We investigated the prevalence of antibiotic resistant bacteria (ARB) and the spread of ARGs in intensive bullfrog (Rana catesbeiana) farms in the Shantou area of China. Antibiotic susceptibilities of 361 strains, combined with microbiome analyses, revealed Escherichia coli, Edwardsiella tarda, Citrobacter and Klebsiella sp. as prevalent multidrug resistant bacteria on these farms. Whole genome sequencing of 95 ARB identified 250 large plasmids that harbored a wide range of ARGs. Plasmid sequences and sediment metagenomes revealed an abundance of tetA, sul1, and aph(3″)-Ib ARGs. Notably, antibiotic resistance (against 15 antibiotics) highly correlated with plasmid-borne rather than chromosome-borne ARGs. Based on sequence similarities, most plasmids (62%) fell into 32 distinct groups, indicating a potential for horizontal plasmid transfer (HPT) within the frog farm microbiome. HPT was confirmed in inter- and intra-species conjugation experiments. Furthermore, identical mobile ARGs, flanked by mobile genetic elements (MGEs), were found in different locations on the same plasmid, or on different plasmids residing in the same or different hosts. Our results suggest a synergy between MGEs and HPT to facilitate ARGs dissemination in frog farms. Mining public databases retrieved similar plasmids from different bacterial species found in other environmental niches globally. Our findings underscore the importance of HPT in mediating the spread of ARGs in frog farms and other microbiomes of the ecosystem.

During the COVID-19 pandemic, contact tracing was used to identify individuals who had been in contact with a confirmed case so that these contacted individuals could be tested and quarantined to prevent further spread of the SARS-CoV-2 virus. Many countries developed mobile apps to find these contacted individuals faster. We evaluate the epidemiological effectiveness of the Dutch app CoronaMelder, where we measure effectiveness as the reduction of the reproduction number R. To this end, we use a simulation model of SARS-CoV-2 spread and contact tracing, informed by data collected during the study period (December 2020 - March 2021) in the Netherlands. We show that the tracing app caused a clear but small reduction of the reproduction number, and the magnitude of the effect was found to be robust in sensitivity analyses. The app could have been more effective if more people had used it, and if notification of contacts could have been done directly by the user and thus reducing the time intervals between symptom onset and reporting of contacts. The model has two innovative aspects: i) it accounts for the clustered nature of social networks and ii) cases can alert their contacts informally without involvement of health authorities or the tracing app.

Edwardsiella species in aquatic environments exist either as individual planktonic cells or in communal biofilms. These organisms encounter multiple stresses, include changes in salinity, pH, temperature, and nutrients. Pathogenic species such as E. piscicida, can multiply within the fish hosts. Additionally, Edwardsiella species (E. tarda), can carry antibiotic resistance genes (ARGs) on chromosomes and/or plasmids, that can be transmitted to the microbiome via horizontal gene transfer. E. tarda serves as a core in the aquatic resistome. Edwardsiela uses molecular switches (RpoS and EsrB) to control gene expression for survival in different environments. We speculate that free-living Edwardsiella can transition to host-living and vice versa, using similar molecular switches. Understanding such transitions can help us understand how other similar aquatic bacteria switch from free-living to become pathogens. This knowledge can be used to devise ways to slow down the spread of ARGs and prevent disease outbreaks in aquaculture and clinical settings.

High vaccination coverage is considered to be key in dealing with the coronavirus disease (COVID-19) pandemic. However, vaccine hesitancy can limit uptake. We examined the specific coronavirus beliefs that persons have regarding COVID-19 and COVID-19 vaccines and to what extent these beliefs explain COVID-19 vaccination intentions. We conducted a survey among 4,033 residents of the Netherlands that examined COVID-19 vaccination intentions and various beliefs. Random forest regression analysis explained 76% of the variance in vaccination intentions. The strongest determinant in the model was the belief the COVID-19 crisis will only end if many persons get vaccinated. Other strong determinants were beliefs about safety of vaccines, specifically in relation to vaccine development and approval process; (social) benefits of vaccination; social norms regarding vaccination behavior; and effectiveness of vaccines. We propose to address these specific beliefs in communications about COVID-19 vaccinations to stimulate vaccine uptake.

Introduction A substantial proportion of individuals infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), report persisting symptoms weeks and months following acute infection. Estimates on prevalence vary due to differences in study designs, populations, heterogeneity of symptoms and the way symptoms are measured. Common symptoms include fatigue, cognitive impairment and dyspnoea. However, knowledge regarding the nature and risk factors for developing persisting symptoms is still limited. Hence, in this study, we aim to determine the prevalence, severity, risk factors and impact on quality of life of persisting symptoms in the first year following acute SARS-CoV-2 infection.Methods and analysis The LongCOVID-study is both a prospective and retrospective cohort study being conducted in the Netherlands, with a one year follow-up. Participants aged 5 years and above, with self-reported positive or negative tests for SARS-CoV-2 will be included in the study. The primary outcome is the prevalence and severity of persistent symptoms in participants that tested positive for SARS-CoV-2 compared with controls. Symptom severity will be assessed for fatigue (Checklist Individual Strength (CIS subscale fatigue severity)), pain (Rand-36/SF-36 subscale bodily pain), dyspnoea (Medical Research Council (mMRC)) and cognitive impairment (Cognitive Failure Questionnaire (CFQ)). Secondary outcomes include effect of vaccination prior to infection on persistent symptoms, loss of health-related quality of life (HRQoL) and risk factors for persisting symptoms following infection with SARS-CoV-2.Ethics and dissemination The Utrecht Medical Ethics Committee (METC) declared in February 2021 that the Medical Research Involving Human Subjects Act (WMO) does not apply to this study (METC protocol number 21-124/C). Informed consent is required prior to participation in the study. Results of this study will be submitted for publication in a peer-reviewed journal.

ABSTRACT Assembly of a resistome in parallel with the establishment of a microbial community is not well understood. Germfree models can reveal microbiota interactions and shed light on bacterial colonization and resistance development under antibiotic pressure. In this study, we exposed germfree soil (GS), GS with diluted nontreated soil (DS), and nontreated soil (NS) to various concentrations of tetracycline (TET) in a nongermfree environment for 10 weeks, followed by 2 weeks of exposure to water. High-throughput sequencing was used to profile bacterial communities and antibiotic resistance genes (ARGs) in the soils. The initial bacterial loads were found to shape the profiles of bacterial communities and the resistomes. GS and DS treated with TET and the same soils left untreated had similar profiles, whereas NS showed different profiles. Soils with the same initial bacterial loads had their profiles shifted by TET treatment. Multidrug resistance (MDR) genes were the most abundant ARG types in all soils, with multidrug efflux pump genes being the discriminatory ARGs in GS regardless of different TET treatments and in GS, DS, and NS after TET. Furthermore, MDR genes were significantly enriched by TET treatment. In contrast, tetracycline resistance genes were either absent or low in relative abundance. The family Burkholderiaceae was predominant in all soils (except in NS treated with water) and was positively selected for by TET treatment. Most importantly, Burkholderiaceae were the primary carrier of ARGs, including MDR genes. IMPORTANCE This is the first study to examine how resistomes develop and evolve using GS. GS can be used to study the colonization and establishment of bacterial communities under antibiotic selection. Surprisingly, MDR genes were the main ARGs detected in GS, and TET treatments did not positively select for specific tetracycline resistance genes. Additionally, Burkholderiaceae were the key bacterial hosts for MDR genes in the current GS model under the conditions investigated. These results show that the family Burkholderiaceae underpins the development of resistome and serves as a source of ARGs. The ease of establishment of Burkholderiaceae and MDR genes in soils has serious implications for human health, since these bacteria are versatile and ubiquitous in the environment.

For the control of COVID-19, vaccination programmes provide a long-term solution. The amount of available vaccines is often limited, and thus it is crucial to determine the allocation strategy. While mathematical modelling approaches have been used to find an optimal distribution of vaccines, there is an excessively large number of possible allocation schemes to be simulated. Here, we propose an algorithm to find a near-optimal allocation scheme given an intervention objective such as minimization of new infections, hospitalizations, or deaths, where multiple vaccines are available. The proposed principle for allocating vaccines is to target subgroups with the largest reduction in the outcome of interest. We use an approximation method to reconstruct the age-specific transmission intensity (the next generation matrix), and express the expected impact of vaccinating each subgroup in terms of the observed incidence of infection and force of infection. The proposed approach is firstly evaluated with a simulated epidemic and then applied to the epidemiological data on COVID-19 in the Netherlands. Our results reveal how the optimal allocation depends on the objective of infection control. In the case of COVID-19, if we wish to minimize deaths, the optimal allocation strategy is not efficient for minimizing other outcomes, such as infections. In simulated epidemics, an allocation strategy optimized for an outcome outperforms other strategies such as the allocation from young to old, from old to young, and at random. Our simulations clarify that the current policy in the Netherlands (i.e., allocation from old to young) was concordant with the allocation scheme that minimizes deaths. The proposed method provides an optimal allocation scheme, given routine surveillance data that reflect ongoing transmissions. This approach to allocation is useful for providing plausible simulation scenarios for complex models, which give a more robust basis to determine intervention strategies.

Edwardsiella piscicida is an Enterobacteriaceae that is abundant in water and causes food and waterborne infections in fish, animals, and humans. The bacterium causes Edwardsiellosis in farmed fish and can lead to severe economic losses in aquaculture worldwide. E. piscicida is an intracellular pathogen that can also cause systemic infection. Type III and type VI secretion systems are the bacterium’s most lethal weapons against host defenses. It also possesses multi-antibiotic resistant genes and is selected and enriched in the environment due to the overuse of antibiotics. Therefore, the bacterium has great potential to contribute to the evolution of the resistome. All these properties have made this bacterium a perfect model to study bacteria virulence mechanisms and the spread of antimicrobial genes in the environment. We summarize recent advance in E. piscicida biology and provide insights into future research in virulence mechanisms, vaccine development and novel therapeutics.

The structure of the sexual network of a population plays an essential role in the transmission of HIV. Concurrent partnerships, i.e. partnerships that overlap in time, are important in determining this network structure. Men and women may differ in their concurrent behavior, e.g. in the case of polygyny where women are monogamous while men may have concurrent partnerships. Polygyny has been shown empirically to be negatively associated with HIV prevalence, but the epidemiological impacts of other forms of gender-asymmetric concurrency have not been formally explored. Here we investigate how gender asymmetry in concurrency, including polygyny, can affect the disease dynamics. We use a model for a dynamic network where individuals may have concurrent partners. The maximum possible number of simultaneous partnerships can differ for men and women, e.g. in the case of polygyny. We control for mean partnership duration, mean lifetime number of partners, mean degree, and sexually active lifespan. We assess the effects of gender asymmetry in concurrency on two epidemic phase quantities (R0 and the contribution of the acute HIV stage to R0) and on the endemic HIV prevalence. We find that gender asymmetry in concurrent partnerships is associated with lower levels of all three epidemiological quantities, especially in the polygynous case. This effect on disease transmission can be attributed to changes in network structure, where increasing asymmetry leads to decreasing network connectivity.

ObjectivesSince 2017, the Public Health Agency of Sweden recommends that pre-exposure prophylaxis (PrEP) for HIV should be offered to high-risk individuals, in particular to men who have sex with men (MSM). The objective of this study is to develop a mathematical model investigating the effect of introducing PrEP to MSM in Sweden.DesignA pair formation model, including steady and casual sex partners, is developed to study the impact of introducing PrEP. Two groups are included in the model: sexually high active MSM and sexually low active MSM. Three mixing assumptions between the groups are considered.SettingA gay-friendly MSM HIV/sexually transmitted infection testing clinic in Stockholm, Sweden. This clinic started offering PrEP to MSM in October 2018.ParticipantsThe model is calibrated according to detailed sexual behaviour data gathered in 2015 among 403 MSM.ResultsBy targeting sexually high active MSM, a PrEP coverage of 3.5% of the MSM population (10% of all high actives) would result in the long-term HIV prevalence to drop considerably (close to 0%). While targeting only low actives would require a PrEP coverage of 35% for a similar reduction. The main effect of PrEP is the reduced susceptibility, whereas the increased HIV testing rate (every third month) among PrEP users plays a lesser role.ConclusionsTo create a multifaceted picture of the effects of interventions against HIV, we need models that include the different stages of HIV infection and real-world data on detailed sexual behaviour to calibrate the mathematical models. Our findings conclude that targeting HIV high-risk individuals, within HIV risk populations such as MSM, with PrEP programmes could greatly decrease the long-term HIV prevalence in Sweden. Therefore, risk stratification of individuals is of importance in PrEP implementation programmes, to ensure optimising the effect and cost-effectiveness of such programmes.

ABSTRACT Transposon insertion sequencing (TIS) is a powerful high-throughput genetic technique that is transforming functional genomics in prokaryotes, because it enables genome-wide mapping of the determinants of fitness. However, current approaches for analyzing TIS data assume that selective pressures are constant over time and thus do not yield information regarding changes in the genetic requirements for growth in dynamic environments (e.g., during infection). Here, we describe structured analysis of TIS data collected as a time series, termed pattern analysis of conditional essentiality (PACE). From a temporal series of TIS data, PACE derives a quantitative assessment of each mutant’s fitness over the course of an experiment and identifies mutants with related fitness profiles. In so doing, PACE circumvents major limitations of existing methodologies, specifically the need for artificial effect size thresholds and enumeration of bacterial population expansion. We used PACE to analyze TIS samples of Edwardsiella piscicida (a fish pathogen) collected over a 2-week infection period from a natural host (the flatfish turbot). PACE uncovered more genes that affect E. piscicida’s fitness in vivo than were detected using a cutoff at a terminal sampling point, and it identified subpopulations of mutants with distinct fitness profiles, one of which informed the design of new live vaccine candidates. Overall, PACE enables efficient mining of time series TIS data and enhances the power and sensitivity of TIS-based analyses. IMPORTANCE Transposon insertion sequencing (TIS) enables genome-wide mapping of the genetic determinants of fitness, typically based on observations at a single sampling point. Here, we move beyond analysis of endpoint TIS data to create a framework for analysis of time series TIS data, termed pattern analysis of conditional essentiality (PACE). We applied PACE to identify genes that contribute to colonization of a natural host by the fish pathogen Edwardsiella piscicida. PACE uncovered more genes that affect E. piscicida’s fitness in vivo than were detected using a terminal sampling point, and its clustering of mutants with related fitness profiles informed design of new live vaccine candidates. PACE yields insights into patterns of fitness dynamics and circumvents major limitations of existing methodologies. Finally, the PACE method should be applicable to additional “omic” time series data, including screens based on clustered regularly interspaced short palindromic repeats with Cas9 (CRISPR/Cas9).

The type VI secretion system (T6SS) of pathogenic bacteria plays important roles in both virulence and inter-bacterial competitions. The effectors of T6SS are presumed to be transported either by attaching to the tip protein or by interacting with HcpI (haemolysin corregulated protein 1). In Edwardsiella tarda PPD130/91, the T6SS secreted protein EvpP (E. tarda virulent protein P) is found to be essential for virulence and directly interacts with EvpC (Hcp-like), suggesting that it could be a potential effector. Using limited protease digestion, nuclear magnetic resonance heteronuclear Nuclear Overhauser Effects, and hydrogen-deuterium exchange mass spectrometry, we confirmed that the dimeric EvpP (40 kDa) contains a substantial proportion (40%) of disordered regions but still maintains an ordered and folded core domain. We show that an N-terminal, 10-kDa, protease-resistant fragment in EvpP connects to a shorter, 4-kDa protease-resistant fragment through a highly flexible region, which is followed by another disordered region at the C-terminus. Within this C-terminal disordered region, residues Pro143 to Ile168 are essential for its interaction with EvpC. Unlike the highly unfolded T3SS effector, which has a lower molecular weight and is maintained in an unfolded conformation with a dedicated chaperone, the T6SS effector seems to be relatively larger, folded but partially disordered and uses HcpI as a chaperone.

BackgroundThe putative needle complex subunit AscF forms a ternary complex with the chaperones AscE and AscG in the type III secretion system of Aeromonas hydrophila so as to avoid premature assembly. Previously, we demonstrated that the C-terminal region of AscG (residues 62-116) in the hetero-molecular chaperone, AscE-AscG, is disordered and susceptible to limited protease digestion.Methodology/principal findingsHere, we report the crystal structure of the ordered AscG(1-61) region in complex with AscE at 2.4 Å resolution. Helices α2 and α3 of AscE in the AscE-AscG(1-61) complex assumes a helix-turn-helix conformation in an anti-parallel fashion similar to that in apo AscE. However, in the presence of AscG, an additional N-terminal helix α1 in AscE (residues 4-12) is observed. PscG or YscG in the crystal structures of PscE-PscF-PscG or YscE-YscF-YscG, respectively, assumes a typical tetratricopeptide repeat (TPR) fold with three TPR repeats and one C-terminal capping helix. By comparison, AscG in AscE-AscG(1-61) comprises three anti-parallel helices that resembles the N-terminal TPR repeats in the corresponding region of PscG or YscG in PscE-PscF-PscG or YscE-YscF-YscG. Thermal denaturation of AscE-AscG and AscE-AscG(1-61) complexes demonstrates that the C-terminal disordered region does not contribute to the thermal stability of the overall complex.Conclusion/significanceThe N-terminal region of the AscG in the AscE-AscG complex is ordered and assumes a structure similar to those in the corresponding regions of PscE-PscG-PscF or YscE-YscF-YscG complexes. While the C-terminal region of AscG in the AscE-AscG complex is disordered and will assume its structure only in the presence of the substrate AscF. We hypothesize that AscE act as a chaperone of the chaperone to keep AscG in a stable but partially disordered state for interaction with AscF.

The recently identified type VI secretion system (T6SS) is implicated in the virulence of many gram-negative bacteria. Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also gastro- and extra-intestinal infections in humans. The E. tardavirulent protein (EVP) gene cluster encodes a conserved T6SS which contains 16 open reading frames. EvpC is one of the three major EVP secreted proteins and shares high sequence similarity with Hcp1, a key T6SS virulence factor from Pseudomonas aeruginosa. EvpC contributes to the virulence of E. tarda by playing an essential role in functional T6SS. Here, we report the crystal structure of EvpC from E. tarda PPD130/91 at a 2.8 Å resolution, along with functional studies of the protein. EvpC has a β-barrel domain with extended loops. The β-barrel consists of 11 anti-parallel β-strands with an α-helix located on one side. In solution, EvpC exists as a dimer at low concentration and as a hexamer at higher concentration. In the crystal, the symmetry related EvpC molecules form hexameric rings which stack together to form a tube similar to Hcp1. Structure based mutagenesis revealed that N-terminal negatively charged residues, Asp4, Glu15 and Glu26, and C-terminal positively charged residues, Lys161, Lys162 and Lys163, played crucial roles in the secretion of EvpC. Moreover, the localization study indicates the presence of wild type EvpC in cytoplasm, periplasm and secreted fractions, whereas the N-terminal and C-terminal mutants were found mostly in the periplasmic region and was completely absent in the secreted fraction. Results reported here provide insight into the structure, assembly and function of EvpC. Further, these findings can be extended to other EvpC homologs for understanding the mechanism of T6SS and targeting T6SS mediated virulence in gram-negative pathogens.

Enterohemorrhagic Escherichia coli (EHEC) is a common cause of severe hemorrhagic colitis. EHEC's virulence is dependent upon a type III secretion system (TTSS) encoded by 41 genes. These genes are organized in several operons clustered in the locus of enterocyte effacement. Most of the locus of enterocyte effacement genes, including grlA and grlR, are positively regulated by Ler, and Ler expression is positively and negatively modulated by GrlA and GrlR, respectively. However, the molecular basis for the GrlA and GrlR activity is still elusive. We have determined the crystal structure of GrlR at 1.9 A resolution. It consists of a typical beta-barrel fold with eight beta-strands containing an internal hydrophobic cavity and a plug-like loop on one side of the barrel. Strong hydrophobic interactions between the two beta-barrels maintain the dimeric architecture of GrlR. Furthermore, a unique surface-exposed EDED (Glu-Asp-Glu-Asp) motif is identified to be critical for GrlA-GrlR interaction and for the repressive activity of GrlR. This study contributes a novel molecular insight into the mechanism of GrlR function.

High vaccination coverage is considered to be key in dealing with the coronavirus disease (COVID-19) pandemic. However, vaccine hesitancy can limit uptake. We examined the specific coronavirus beliefs that persons have regarding COVID-19 and COVID-19 vaccines and to what extent these beliefs explain COVID-19 vaccination intentions. We conducted a survey among 4,033 residents of the Netherlands that examined COVID-19 vaccination intentions and various beliefs. Random forest regression analysis explained 76% of the variance in vaccination intentions. The strongest determinant in the model was the belief the COVID-19 crisis will only end if many persons get vaccinated. Other strong determinants were beliefs about safety of vaccines, specifically in relation to vaccine development and approval process; (social) benefits of vaccination; social norms regarding vaccination behavior; and effectiveness of vaccines. We propose to address these specific beliefs in communications about COVID-19 vaccinations to stimulate vaccine uptake. [ABSTRACT FROM AUTHOR]

Objectives Since 2017, the Public Health Agency of Sweden recommends that pre-exposure prophylaxis (PrEP) for HIV should be offered to high-risk individuals, in particular to men who have sex with men (MSM). The objective of this study is to develop a mathematical model investigating the effect of introducing PrEP to MSM in Sweden. Design A pair formation model, including steady and casual sex partners, is developed to study the impact of introducing PrEP. Two groups are included in the model: sexually high active MSM and sexually low active MSM. Three mixing assumptions between the groups are considered. Setting A gay-friendly MSM HIV/sexually transmitted infection testing clinic in Stockholm, Sweden. This clinic started offering PrEP to MSM in October 2018. Participants The model is calibrated according to detailed sexual behaviour data gathered in 2015 among 403 MSM. Results By targeting sexually high active MSM, a PrEP coverage of 3.5% of the MSM population (10% of all high actives) would result in the long-term HIV prevalence to drop considerably (close to 0%). While targeting only low actives would require a PrEP coverage of 35% for a similar reduction. The main effect of PrEP is the reduced susceptibility, whereas the increased HIV testing rate (every third month) among PrEP users plays a lesser role. Conclusions To create a multifaceted picture of the effects of interventions against HIV, we need models that include the different stages of HIV infection and real-world data on detailed sexual behaviour to calibrate the mathematical models. Our findings conclude that targeting HIV high-risk individuals, within HIV risk populations such as MSM, with PrEP programmes could greatly decrease the long-term HIV prevalence in Sweden. Therefore, risk stratification of individuals is of importance in PrEP implementation programmes, to ensure optimising the effect and cost-effectiveness of such programmes. [ABSTRACT FROM AUTHOR]

Background: Many gram-negative bacteria use type III secretion systems (T3SSs) to translocate effector proteins into host cells. T3SS effectors can give some bacteria a competitive edge over others within the same environment and can help bacteria to invade the host cells and allow them to multiply rapidly within the host. Therefore, developing efficient methods to identify effectors scattered in bacterial genomes can lead to a better understanding of host-pathogen interactions and ultimately to important medical and biotechnological applications. Results: We used 21 genomic and proteomic attributes to create a precise and reliable T3SS effector prediction method called Genome Search for Effectors Tool (GenSET). Five machine learning algorithms were trained on effectors selected from different organisms and a trained (voting) algorithm was then applied to identify other effectors present in the genome testing sets from the same (GenSET Phase 1) or different (GenSET Phase 2) organism. Although a select group of attributes that included the codon adaptation index, probability of expression in inclusion bodies, N-terminal disorder, and G + C content (filtered) were better at discriminating between positive and negative sets, algorithm performance was better when all 21 attributes (unfiltered) were used. Performance scores (sensitivity, specificity and area under the curve) from GenSET Phase 1 were better than those reported for six published methods. More importantly, GenSET Phase 1 ranked more known effectors (70.3%) in the top 40 ranked proteins and predicted 10-80% more effectors than three available programs in three of the four organisms tested. GenSET Phase 2 predicted 43.8% effectors in the top 40 ranked proteins when tested on four related or unrelated organisms. The lower prediction rates from GenSET Phase 2 may be due to the presence of different translocation signals in effectors from different T3SS families. Conclusions: The species-specific GenSET Phase 1 method offers an alternative approach to T3SS effector prediction that can be used with other published programs to improve effector predictions. Additionally, our approach can be applied to predict effectors of other secretion systems as long as these effectors have translocation signals embedded in their sequences. [ABSTRACT FROM AUTHOR]

Background: The putative needle complex subunit AscF forms a ternary complex with the chaperones AscE and AscG in the type III secretion system of Aeromonas hydrophila so as to avoid premature assembly. Previously, we demonstrated that the C-terminal region of AscG (residues 62-116) in the hetero-molecular chaperone, AscE-AscG, is disordered and susceptible to limited protease digestion. Methodology/Principal Findings: Here, we report the crystal structure of the ordered AscG1-61 region in complex with AscE at 2.4 Å resolution. Helices α2 and α3 of AscE in the AscE-AscG1-61 complex assumes a helix-turn-helix conformation in an anti-parallel fashion similar to that in apo AscE. However, in the presence of AscG, an additional N-terminal helix α1 in AscE (residues 4-12) is observed. PscG or YscG in the crystal structures of PscE-PscF-PscG or YscE-YscF-YscG, respectively, assumes a typical tetratricopeptide repeat (TPR) fold with three TPR repeats and one C-terminal capping helix. By comparison, AscG in AscE-AscG1-61 comprises three anti-parallel helices that resembles the N-terminal TPR repeats in the corresponding region of PscG or YscG in PscE-PscF-PscG or YscE-YscF-YscG. Thermal denaturation of AscE-AscG and AscEAscG1- 61 complexes demonstrates that the C-terminal disordered region does not contribute to the thermal stability of the overall complex. Conclusion/Significance: The N-terminal region of the AscG in the AscE-AscG complex is ordered and assumes a structure similar to those in the corresponding regions of PscE-PscG-PscF or YscE-YscF-YscG complexes. While the C-terminal region of AscG in the AscE-AscG complex is disordered and will assume its structure only in the presence of the substrate AscF. We hypothesize that AscE act as a chaperone of the chaperone to keep AscG in a stable but partially disordered state for interaction with AscF. [ABSTRACT FROM AUTHOR]

The recently identified type VI secretion system (T6SS) is implicated in the virulence of many Gram-negative bacteria. Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also gastro- and extra-intestinal infections in humans. The E. tarda virulent protein (EVP) gene cluster encodes a conserved T6SS which contains 16 open reading frames. EvpC is one of the three major EVP secreted proteins and shares high sequence similarity with Hcp1, a key T6SS virulence factor from Pseudomonas aeruginosa. EvpC contributes to the virulence of E. tarda by playing an essential role in functional T6SS. Here, we report the crystal structure of EvpC from E. tarda PPD130/91 at a 2.8 Å resolution, along with functional studies of the protein. EvpC has a β-barrel domain with extended loops. The β-barrel consists of 11 anti-parallel β-strands with an a-helix located on one side. In solution, EvpC exists as a dimer at low concentration and as a hexamer at higher concentration. In the crystal, the symmetry related EvpC molecules form hexameric rings which stack together to form a tube similar to Hcp1. Structure based mutagenesis revealed that N-terminal negatively charged residues, Asp4, Glu15 and Glu26, and C-terminal positively charged residues, Lys161, Lys162 and Lys163, played crucial roles in the secretion of EvpC. Moreover, the localization study indicates the presence of wild type EvpC in cytoplasm, periplasm and secreted fractions, whereas the N-terminal and C-terminal mutants were found mostly in the periplasmic region and was completely absent in the secreted fraction. Results reported here provide insight into the structure, assembly and function of EvpC. Further, these findings can be extended to other EvpC homologs for understanding the mechanism of T6SS and targeting T6SS mediated virulence in Gram-negative pathogens. [ABSTRACT FROM AUTHOR]

Bacterial pathogens use different protein secretion systems to deliver virulence factors. Recently, a novel secretion system was discovered in several Gram-negative bacterial pathogens, and was designated as the type VI secretion system (T6SS). In Edwardsiella tarda, a partial . tarda irulent rotein (EVP) gene cluster was implicated in protein secretion. Here, we identified the entire EVP cluster as a T6SS and two additional secreted proteins (EvpI, a homologue of VgrG, and EvpP) were found. We systematically mutagenized all the 16 EVP genes and found that the secretion of EvpP was dependent on 13 EVP proteins including EvpC (a homologue of Hcp) and EvpI but not EvpD and EvpJ. All EVP mutants except Δ evpD were attenuated in blue gourami fish. The 16 EVP proteins can be grouped according to their functions and cellular locations. The first group comprises 11 non-secreted and possibly intracellular apparatus proteins. Among them, EvpO, a putative ATPase which contained a Walker A motif, showed possible interactions with three EVP proteins (EvpA, EvpL and EvpN). The second group includes three secreted proteins (EvpC, EvpI and EvpP). The secretion of EvpC and EvpI is mutually dependent, and they are required for the secretion of EvpP. The interaction between EvpC and EvpP was demonstrated. Lastly, two proteins (EvpD and EvpJ) are not required for the T6SS-dependent secretion. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) is a common cause of severe hemorrhagic colitis. EHEC's virulence is dependent upon a type III secretion system (TTSS) encoded by 41 genes. These genes are organized in several operons clustered in the locus of enterocyte effacement. Most of the locus of enterocyte effacement genes, including grlA and grlR, are positively regulated by Ler, and Ler expression is positively and negatively modulated by GrlA and GrlR, respectively. However, the molecular basis for the GrlA and GrlR activity is still elusive. We have determined the crystal structure of GrlR at 1.9 Å resolution. It consists of a typical β-barrel fold with eight β-strands containing an internal hydrophobic cavity and a plug-like loop on one side of the barrel. Strong hydrophobic interactions between the two β-barrels maintain the dimeric architecture of GrlR. Furthermore, a unique surface-exposed EDED (Glu-Asp-Glu-Asp) motif is identified to be critical for GrlA-GrlR interaction and for the repressive activity of GrlR. This study contributes a novel molecular insight into the mechanism of GrlR function. [ABSTRACT FROM AUTHOR]

Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastro- and extraintestinal infections in humans. Using a combination of comparative proteomics and Tn phoA mutagenesis, we have identified five proteins that may contribute to E. tarda PPD130/91 pathogenesis. Lowered protein secretion, impaired autoaggregation and the absence of six proteins were observed only in three highly attenuated mutants when cultured in Dulbecco's modified eagle medium (DMEM). Five out of six proteins could be identified by their mass spectra. Three proteins were identified as putative effector proteins (EseB, EseC and EseD) that are homologous to SseB, SseC and SseD of a type III secretion system (TTSS) in Salmonella species. The other two were EvpA and EvpC, homologous to Eip20 and Eip18 in Edwardsiella ictaluri. The complete sequencing and homology studies of evpA–H indicate that similar gene clusters are widely distributed in other pathogens such as Escherichia, Salmonella, Vibrio and Yersinia species with unknown functions. Insertional inactivation and deletion of evpB or evpC led to lower replication rates in gourami phagocytes, and reduced protein secretion and virulence in blue gourami. Complementation of these deletion mutants showed partial recovery in the above three phenotypes, indicating that these genes are vital for E. tarda pathogenesis. The transport of the EvpC protein may not use the TTSS in E. tarda. The expression of EvpA and EvpC as well as EseB, EseC and EseD was temperature dependent (suppressed at 37°C), and disruption of esrB affected their expression. The present study identifies two possible secretion systems (TTSS and Evp) that are vital for E. tarda pathogenesis. [ABSTRACT FROM AUTHOR]

Salmonella species are facultative intracellular pathogens that invade epithelial cells and reside within lysosomal membrane glycoprotein (lgp)-containing vacuoles. Coincident with the onset of bacterial replication inside these vacuoles, Salmonella induce the formation of stable Igp-containing filamentous structures that connect with the Salmonella-containing vacuoles. Salmonella typhimurium SL1344::Tn10dCm mutant strains unable to induce these structures were isolated. Ail contained insertions within a novel S̱almonella ḻnduced f̱ilament gene A (sifA). sifA is present only in Salmonella species and encodes a protein with a predicted molecular mass of 38 kDa and an apparent molecular mass of 35 kDa. sifA is flanked by ∼300 base pairs, and sifA and its flanking DNA show no homology to sequences in DNA databases. sifA is located within the potABCD operon, a housekeeping locus involved in periplasmic transport of polyamines. Fourteen-base-pair direct repeats mark the probable site of integration of sifA and its flanking DNA at the 3′ end of potB. sifA and its flanking DNA have a significantly reduced G+C content (41%) when compared with the potABCD operon (51%) and the Salmonella genome (52-54%). Deletion mutant strains in sifA or in the downstream potC were constructed. ΔsifA does not produce Salmonella-induced filaments in epithelial cells, and is attenuated in mice. ΔpofC produces Salmonella-induced filaments in epithelial cells, and was fully virulent. Collectively, these results suggest that sifA arose by horizontal gene transfer into Salmonella and its product is involved in a virulence-associated intracellular phenotype related to Salmonella-induced filament formation. [ABSTRACT FROM AUTHOR]

Edwardsiella tarda causes haemorrhagic septicaemia in fish and gastro- and extra-intestinal infections in animals including humans. Resistance to phagocyte-mediated killing is one of the virulence factors of Ed. tarda. The authors' previous studies using TnphoA transposon mutagenesis indicated that katB mutants derived from the strain PPD130/91 are at least 1·6 log higher in LD[sub 50] values than the wild-type strain. These findings suggest the involvement of catalase (KatB) in Ed. tarda pathogenesis. In this study, experiments were conducted to characterize the contribution of KatB to Ed. tarda infection. Zymographic analyses indicated that the 22 Ed. tarda strains examined expressed three different types of catalase-peroxidases (Kat1-3) based on their mobility in non-denaturing polyacrylamide gels. KatB (Kat1), the major catalase enzyme, was expressed in eight out of 22 Ed. tarda strains, and was commonly found in virulent strains except AL9379. AL9379 has a mutated katB, which has a base substitution and a deletion that translate into stop codons in the catalase gene. KatB produced by PPD130/91 was located in both periplasmic and cytoplasmic fractions and was constitutively expressed in various growth phases. Kinetics studies indicated that the catalase provided resistance to H[sub 2]O[sub 2]- and phagocyte-mediated killing. Infection kinetics studies of katB mutant 34 in gourami fish demonstrated its inability to survive and replicate in phagocyte-rich organs and this prevented the dissemination of infections when compared to the wild-type. Complementation of catalase mutants restored the production of catalase, and led to an increase in the resistance to H[sub 2]O[sub 2]- and phagocyte-mediated killing, and a decrease in LD[sub 50] values. This study has identified and characterized a major catalase gene (katB) that is required for resistance to H[sub 2]O[sub 2]- and phagocyte-mediated killing in Ed. tarda. The results also suggest that catalase may play a role as a virulence factor in Ed. tarda pathogenesis. [ABSTRACT FROM AUTHOR]

The PhoP-PhoQ two-component system is commonly used by bacteria to sense environmental factors. Here we show that the PhoP-PhoQ system of Edwardsiella tarda detects changes in environmental temperature and Mg2+ concentration as well as regulates the type III and VI secretion systems through direct activation of esrB. Protein secretion is activated from 23 to 35 °C or at low Mg2+ concentrations, but it is suppressed at or below 20 °C, at or above 37 °C, or at high Mg2+ concentrations. The effects of temperature and Mg2+ concentration are additive. The PhoQ sensor domain has a low Tm of 37.9 °C, and it detects temperatures through a conformational change of its secondary structure. Mutation of specific Pro or Thr residues increased the stability of the PhoQ sensor drastically, altering its temperature-sensing ability. The PhoQ sensor detects Mg2+ concentration through the direct binding of Mg2+ to a cluster of acidic residues (DDDSAD) and through changes that likely affect its tertiary structure. Here, we describe for the first time the use of PhoP-PhoQ as a temperature sensor for bacterial virulence control. [ABSTRACT FROM AUTHOR]

The article presents a study on EscA as a protein necessary in the accumulation and proper secretion of EseC, another translocon component. It provides the methods used in the investigation, involving bacteria strains, plasmids, culture media and growth conditions and construction of in-frame deletion mutants of Edwardsiella tarda. The results suggested that EscA serves as a particular chaperone for EseC and stimulates the virulence of Edwardsiella tarda, a significant Gram-negative enteric pathogen harming both animals and humans.

The article presents microbiological research into the role of the EscC open reading frame and small protein as a chaperone for the Edwardsiella tarda type III secretion system (T3SS) putative translocon components EseB and EseD. EscC is located next to the T3SS gene cluster translocators. Results indicate EscC mutation did not affect the transcription of EseB.