We present the investigation of a Burkholderia pseudomallei strain isolated from the urine of a 56-year-old male returning from a recreational trip to Thailand. The patient initially presented with fever and chills, and lobar as well as bladder and prostate involvement was demonstrated on imaging. Treatment first involved trimethoprim-sulfamethoxazole, the backbone of melioidosis therapy, but was discontinued upon the discovery of resistance to the drug through antimicrobial sensitivity testing via broth microdilution. Further analysis included genomic sequencing, immunotyping, and phenotypic assessment, including virulence testing in an animal model. Results indicated that this strain is a distinct, novel variant with antigenic similarity to other Thai strains displaying high virulence, with a significantly low murine intranasal lethal dose 50% inoculum. The emergence of such a strain, particularly if prevalent in tourist destinations in Thailand, could pose a substantial public health risk, highlighting the need for ongoing vigilance among infectious disease specialists and clinical microbiologists.

Objective Burkholderia pseudomallei, the etiological cause of melioidosis, is a soil saprophyte endemic in South-East Asia, where it constitutes a public health concern of high-priority. Melioidosis cases are sporadically identified in nonendemic areas, usually associated with travelers or import of goods from endemic regions. Due to extensive intercontinental traveling and the anticipated climate change-associated alterations of the soil bacterial flora, there is an increasing concern for inadvertent establishment of novel endemic areas, which may expand the global burden of melioidosis. Rapid diagnosis, isolation and characterization of B. pseudomallei isolates is therefore of utmost importance particularly in non-endemic locations. Data description We report the genome sequences of two novel clinical isolates (MWH2021 and MST2022) of B. pseudomallei identified in distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The data includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of 131 B. pseudomallei strains documented in the NCBI database. Inspection of the genomic data revealed the presence or absence of loci encoding for several documented virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or chronic melioidosis established that both strains belong to the highly virulent class of B. pseudomalleii.

The global spread of SARS-CoV-2 led to major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the virus pathogenesis, and facilitate the development of novel therapeutics. Here, employing a genome-scale CRISPR screen, we provide a comprehensive data-set of cellular factors that are exploited by wild type SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. We identified several host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination, Heparan sulfate biogenesis and host phosphatidylglycerol biosynthesis. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential proviral gene for all variants inspected. We show that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals shows elevated levels of GATA6, suggesting a role in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and inhibition of viral infectivity. Overall, we show GATA6 may represent a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.

HLA transgenic mice are instrumental for evaluation of human-specific immune responses to viral infection. Mice do not develop COVID-19 upon infection with SARS-CoV-2 due to the strict tropism of the virus to the human ACE2 receptor. The aim of the current study was the implementation of an adenovirus-mediated infection protocol for human ACE2 expression in HLA transgenic mice. Transient pulmonary expression of the human ACE2 receptor in these mice results in their sensitisation to SARS-CoV-2 infection, consequently providing a valuable animal model for COVID-19. Infection results in a transient loss in body weight starting 3 days post-infection, reaching 20–30% loss of weight at day 7 and full recovery at days 11–13 post-infection. The evolution of the disease revealed high reproducibility and very low variability among individual mice. The method was implemented in two different strains of HLA immunized mice. Infected animals developed strong protective humoral and cellular immune responses specific to the viral spike-protein, strictly depending on the adenovirus-mediated human ACE2 expression. Convalescent animals were protected against a subsequent re-infection with SARS-CoV-2, demonstrating that the model may be applied for assessment of efficacy of anti-viral immune responses.

The global spread of SARS-CoV-2 led to the most challenging pandemic in this century, posing major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SASR-CoV-2 can provide insights into the virus pathogenesis, and facilitates the development of novel broad-spectrum host-directed therapeutics. Here, employing genome-scale CRISPR screens, we provide a comprehensive data-set of cellular factors that are exploited by WT-SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. These screens identified known and novel host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination and Heparan sulfate biogenesis. In addition, the host phosphatidylglycerol biosynthesis processes appeared to have major anti-viral functions. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant, providing a possible explanation for the increased infectivity of this variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential pro-viral gene for all variants inspected. We revealed that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals showed an elevated level of GATA6, indicating the important role GATA6 may be playing in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and consequently to inhibition of the viral infectivity. Overall, we show GATA6 represents a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.

The COVID-19 pandemic led to development of mRNA vaccines, which became a leading anti-SARS-CoV-2 immunization platform. Preclinical studies are limited to infection-prone animals such as hamsters and monkeys in which protective efficacy of vaccines cannot be fully appreciated. We recently reported a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). BALB/c mice demonstrated specific immunologic responses following RBD-hFc mRNA vaccination. Now, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model. Administration of an RBD-hFc mRNA vaccine to K18-hACE2 mice resulted in robust humoral responses comprising binding and neutralizing antibodies. In correlation with this response, 70% of vaccinated mice withstood a lethal SARS-CoV-2 dose, while all control animals succumbed to infection. To the best of our knowledge, this is the first nonreplicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD. A subset of the antibodies exert their inhibitory activity by abrogating binding of the RBD to the human ACE2 receptor. The human monoclonal antibodies described here represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.

The high-temperature requirement chaperone/protease (HtrA) is involved in the stress response of the anthrax-causing pathogen Bacillus anthracis . Resilience to oxidative stress is essential for the manifestation of B. anthracis pathogenicity. Here, we announce transcriptome data sets detailing global gene expression in B. anthracis wild-type and htrA -disrupted strains following H 2 O 2 -induced oxidative stress.

We previously demonstrated that the HtrA (High Temperature Requirement A) protease/chaperone active in the quality control of protein synthesis, represents an important virulence determinant of Bacillus anthracis. Virulence attenuation of htrA-disrupted Bacillus anthracis strains was attributed to susceptibility of ΔhtrA strains to stress insults, as evidenced by affected growth under various stress conditions. Here, we report a comparative RNA-seq transcriptomic study generating a database of differentially expressed genes in the B. anthracishtrA-disrupted and wild type parental strains under oxidative stress. The study demonstrates that, apart from protease and chaperone activities, HtrA exerts a regulatory role influencing expression of more than 1000 genes under stress. Functional analysis of groups or individual genes exhibiting strain-specific modulation, evidenced (i) massive downregulation in the ΔhtrA and upregulation in the WT strains of various transcriptional regulators, (ii) downregulation of translation processes in the WT strain, and (iii) downregulation of metal ion binding functions and upregulation of sporulation-associated functions in the ΔhtrA strain. These modulated functions are extensively discussed. Fifteen genes uniquely upregulated in the wild type strain were further interrogated for their modulation in response to other stress regimens. Overexpression of one of these genes, encoding for MazG (a nucleoside triphosphate pyrophosphohydrolase involved in various stress responses in other bacteria), in the ΔhtrA strain resulted in partial alleviation of the H2O2-sensitive phenotype.

Francisella tularensis is a highly infectious intracellular bacterium representing the causative agent of tularemia, a severe disease which requires prompt antibacterial intervention for mitigating its potential high mortality. Inhaled bacteria interact with lung cells belonging to various subpopulations, including those of the epithelium. As of today, the host epithelial response to inhalational infection with F. tularensis is poorly understood. Here, we announce host transcriptome data sets which systematically address the human epithelial response to F. tularensis at different time points postinfection.

We report here the draft genome sequence of Burkholderia pseudomallei MAA2018. This highly virulent strain was isolated in 2018 from the first melioidosis case in Israel associated with recreational travel to Goa, India.

Anthrax is a lethal disease caused by the Gram-positive spore-producing bacterium Bacillus anthracis. We previously demonstrated that disruption of htrA gene, encoding the chaperone/protease HtrABA (High Temperature Requirement A of B. anthracis) results in significant virulence attenuation, despite unaffected ability of ΔhtrA strains (in which the htrA gene was deleted) to synthesize the key anthrax virulence factors: the exotoxins and capsule. B. anthracis ΔhtrA strains exhibited increased sensitivity to stress regimens as well as silencing of the secreted starvation-associated Neutral Protease A (NprA) and down-modulation of the bacterial S-layer. The virulence attenuation associated with disruption of the htrA gene was suggested to reflect the susceptibility of ΔhtrA mutated strains to stress insults encountered in the host indicating that HtrABA represents an important B. anthracis pathogenesis determinant. As all HtrA serine proteases, HtrABA exhibits a protease catalytic domain and a PDZ domain. In the present study we interrogated the relative impact of the proteolytic activity (mediated by the protease domain) and the PDZ domain (presumably necessary for the chaperone activity and/or interaction with substrates) on manifestation of phenotypic characteristics mediated by HtrABA. By inspecting the phenotype exhibited by ΔhtrA strains trans-complemented with either a wild-type, truncated (ΔPDZ), or non-proteolytic form (mutated in the catalytic serine residue) of HtrABA, as well as strains exhibiting modified chromosomal alleles, it is shown that (i) the proteolytic activity of HtrABA is essential for its N-terminal autolysis and subsequent release into the extracellular milieu, while the PDZ domain was dispensable for this process, (ii) the PDZ domain appeared to be dispensable for most of the functions related to stress resilience as well as involvement of HtrABA in assembly of the bacterial S-layer, (iii) conversely, the proteolytic activity but not the PDZ domain, appeared to be dispensable for the role of HtrABA in mediating up-regulation of the extracellular protease NprA under starvation stress, and finally (iv) in a murine model of anthrax, the HtrABA PDZ domain, was dispensable for manifestation of B. anthracis virulence. The unexpected dispensability of the PDZ domain may represent a unique characteristic of HtrABA amongst bacterial serine proteases of the HtrA family.

A previously healthy young man presented with a chronic cavitary pulmonary infection that began while in Goa, India. Burkholderia pseudomallei was cultured from sputum samples. The infection fully resolved after prolonged antibiotic treatment. Other than traveling during the monsoon season, extensive use of well-water for water-pipe smoking of cannabis was identified as a possible risk factor for infection. This is one of the first reports of travel-associated melioidosis from India. Genomic and immunological characterization suggested that the B. pseudomallei isolate collected from the reported case exhibited limited similarity to other B. pseudomallei strains.

Diagnostic identification of pathogens is usually accomplished by isolation of the pathogen or its substances, and should correlate with the time and site of infection. Alternatively, immunoassays such as enzyme-linked immunosorbent assay (ELISA) tests for quantification of serum antibodies are expedient and are usually employed for retrospective diagnostic of a particular infective agent. Here, the potential of cell-based immunoassays for early pathogen detection was evaluated by quantification of specific, antigen-activated, low-frequency IFNγ-secreting cells in mouse spleens following infection with various pathogens. Using enzyme-linked immunospot (ELISPOT) assays, specific responses were observed within 3-6 days following infection with F. tularensis, B. anthracis, Y. pestis, or Influenza virus. Blood samples collected from F. tularensis-infected mice revealed the presence of IFNγ-producing activated cells within one week post infection. When non-human primates were infected with B. anthracis, cellular response was observed in peripheral blood samples as early as five days post infection, 3-5 days earlier than serum antibodies. Finally, the expression pattern of genes in splenocytes of F. tularensis-infected mice was inspected by a transcriptomic approach, enabling the identification of potential host targets for the future development of genetic-based cellular immunoassays. Altogether, the data demonstrate the potential of cell-based immunoassays for early pathogen detection.

We recently reported the development of a novel, next-generation, live attenuated anthrax spore vaccine based on disruption of the htrA (High Temperature Requirement A) gene in the Bacillus anthracis Sterne veterinary vaccine strain. This vaccine exhibited a highly significant decrease in virulence in murine, guinea pig and rabbit animal models yet preserved the protective value of the parental Sterne strain. Here, we report the evaluation of additional mutations in the lef and cya genes, encoding for the toxin components lethal factor (LF) and edema factor (EF), to further attenuate the SterneΔhtrA strain and improve its compatibility for human use. Accordingly, we constructed seven B. anthracis Sterne-derived strains exhibiting different combinations of mutations in the htrA, cya and lef genes. The various strains were indistinguishable in growth in vitro and in their ability to synthesise the protective antigen (PA, necessary for the elicitation of protection). In the sensitive murine model, we observed a gradual increase (ΔhtrA<ΔhtrAΔcya<ΔhtrAΔlef<ΔhtrAΔlefΔcya) in attenuation - up to 108-fold relative to the parental Sterne vaccine strain. Most importantly, all various SterneΔhtrA derivative strains did not differ in their ability to elicit protective immunity in guinea pigs. Immunisation of guinea pigs with a single dose (109 spores) or double doses (>107spores) of the most attenuated triple mutant strain SterneΔhtrAlefMUTΔcya induced a robust immune response, providing complete protection against a subsequent respiratory lethal challenge. Partial protection was observed in animals vaccinated with a double dose of as few as 105spores. Furthermore, protective immune status was maintained in all vaccinated guinea pigs and rabbits for at least 40 and 30weeks, respectively.

Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis . Live attenuated vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔ htrA strain secretes functional anthrax toxins but is 10–10 4 -fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits). In spite of this attenuation, double or even single immunization with SterneΔ htr A spores elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization.

Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules.