Acute Human Infection Research Articles

Development and application of a bacteriophage cocktail for Shigella flexneri biofilm inhibition on the stainless steel surface

Food contamination and biofilm formation by Shigella in food processing facilities are major causes of acute gastrointestinal infection and mortality in humans. Bacteriophages (phages) are promising alternatives to antibiotics in controlling plankton and biofilms in food matrices. This study isolated two novel phages, S2_01 and S2_02, with lytic activity against various Shigella spp. From sewage samples. Transmission electron microscopy revealed that phages S2_01 and S2_02 belonged to the Caudovirales order. On characterizing their lytic ability, phage S2_01 initially exhibited relatively weak antibacterial activity, while phage S2_02 initially displayed rapid antibacterial activity after phage application. A combination of these phages in a 1:9 ratio was selected, as it has been suggested to elicit the most rapid and sustained lysis ability for up to 24 h. It demonstrated lytic activity against various foodborne pathogens, including six Shigella spp. The phage cocktail exhibited biofilm inhibition and disruption abilities of approximately 79.29% and 42.55%, respectively, after 24 h in a 96-well microplate. In addition, inhibition (up to 23.42%) and disruption (up to 19.89%) abilities were also observed on stainless steel surfaces, and plankton growth was also significantly suppressed. Therefore, the phage cocktail formulated in this study displays great potential as a biological control agent in improving food safety against biofilms and plankton.

The loss of the PDIM/PGL virulence lipids causes differential secretion of ESX-1 substrates in Mycobacterium marinum.

The mycobacterial cell envelope is a major virulence determinant in pathogenic mycobacteria. Specific outer lipids play roles in pathogenesis, modulating the immune system and promoting the secretion of virulence factors. ESX-1 (ESAT-6 system-1) is a conserved protein secretion system required for mycobacterial pathogenesis. Previous studies revealed that mycobacterial strains lacking the outer lipid PDIM have impaired ESX-1 function during laboratory growth and infection. The mechanisms underlying changes in ESX-1 function are unknown. We used a proteo-genetic approach to measure phthiocerol dimycocerosate (PDIM)- and phenolic glycolipid (PGL)-dependent protein secretion in M. marinum, a non-tubercular mycobacterial pathogen that causes tuberculosis-like disease in ectothermic animals. Importantly, M. marinum is a well-established model for mycobacterial pathogenesis. Our findings showed that M. marinum strains without PDIM and PGL showed specific, significant reductions in protein secretion compared to the WT and complemented strains. We recently established a hierarchy for the secretion of ESX-1 substrates in four (I-IV) groups. Loss of PDIM differentially impacted secretion of Group III and IV ESX-1 substrates, which are likely the effectors of pathogenesis. Our data suggest that the altered secretion of specific ESX-1 substrates is responsible for the observed ESX-1-related effects in PDIM-deficient strains.IMPORTANCEMycobacterium tuberculosis, the cause of human tuberculosis, killed an estimated 1.3 million people in 2022. Non-tubercular mycobacterial species cause acute and chronic human infections. Understanding how these bacteria cause disease is critical. Lipids in the cell envelope are essential for mycobacteria to interact with the host and promote disease. Strains lacking outer lipids are attenuated for infection, but the reasons are unclear. Our research aims to identify a mechanism for attenuation of mycobacterial strains without the PDIM and PGL outer lipids in M. marinum. These findings will enhance our understanding of the importance of lipids in pathogenesis and how these lipids contribute to other established virulence mechanisms.

Untargeted metabolomics unveiled the role of butanoate metabolism in the development of Pseudomonas aeruginosa hypoxic biofilm.

Pseudomonas aeruginosa is a versatile opportunistic pathogen which causes a variety of acute and chronic human infections, some of which are associated with the biofilm phenotype of the pathogen. We hypothesize that defining the intracellular metabolome of biofilm cells, compared to that of planktonic cells, will elucidate the metabolic pathways and biomarkers indicative of biofilm inception. Disc-shaped stainless-steel coupons (12.7 mm diameter) were employed as a surface for static biofilm establishment. Each disc was immersed in a well, of a 24-well microtiter plate, containing a 1-mL Lysogeny broth (LB) suspension of P. aeruginosa ATCC 9027, a strain known for its biofilm prolificacy. This setup underwent oxygen-depleted incubation at 37°C for 24 hours to yield hypoxic biofilms and the co-existing static planktonic cells. In parallel, another planktonic phenotype of ATCC 9027 was produced in LB under shaking (200 rpm) incubation at 37°C for 24 hours. Planktonic and biofilm cells were harvested, and the intracellular metabolites were subjected to global untargeted metabolomic analysis using LC-MS technology, where small metabolites (below 1.5 kDa) were selected. Data analysis showed the presence of 324 metabolites that differed (p < 0.05) in abundance between planktonic and biofilm cells, whereas 70 metabolites did not vary between these phenotypes (p > 0.05). Correlation, principal components, and partial least square discriminant analyses proved that the biofilm metabolome is distinctly clustered away from that of the two planktonic phenotypes. Based on the functional enrichment analysis, arginine and proline metabolism were enriched in planktonic cells, but butanoate metabolism was enriched in biofilm cells. Key differential metabolites within the butanoate pathway included acetoacetate, 2,3-butandiol, diacetyl, and acetoin, which were highly upregulated in the biofilm compared to the planktonic cells. Exogenous supplementation of acetoin (2 mM), a critical metabolite in butanoate metabolism, augmented biofilm mass, increased the structural integrity and thickness of the biofilm, and maintained the intracellular redox potential by balancing NADH/NAD+ ratio. In conclusion, P. aeruginosa hypoxic biofilm has a specialized metabolic landscape, and butanoate pathway is a metabolic preference and possibly required for promoting planktonic cells to the biofilm state. The butanoate pathway metabolites, particularly acetoin, could serve as markers for biofilm development.

Characterization of a new Pseudomonas aeruginosa Queuovirinae bacteriophage.

The ESKAPEE pathogen Pseudomonas aeruginosa is a common cause of chronic wound and cystic fibrosis lung infections, as well as acute burn and nosocomial infections. Many of these infections are recalcitrant to conventional antibiotic therapies due to both traditional antibiotic resistance mechanisms and antimicrobial tolerance. Recent successes with bacteriophage (phage) therapy to treat chronic human P. aeruginosa infections have led to a renewed interest in isolating and characterizing new P. aeruginosa phages. Here, we isolated and characterized a new lytic phage (termed PIP, pili-infecting phage) capable of infecting P. aeruginosa PA14. PIP is a tailed phage with an icosahedral head and flexible tail containing a genome that is 57,462 bp in length. Phylogenetic analysis reveals that PIP belongs to the subfamily Queuovirinae and genus Nipunavirus but is highly divergent in gene content from known Nipunaviruses. By isolating and characterizing a P. aeruginosa strain that spontaneously evolved resistance to PIP, we show that the receptor for PIP is Type IV pili. In summary, we isolated a new P. aeruginosa phage species with a unique genome, thus increasing the diversity of phages known to infect this important human pathogen.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic human infections. These infections are notoriously difficult to treat due to both antibiotic resistance and antibiotic tolerance. The increasing frequency of antibiotic failure in P. aeruginosa infections has led scientists to explore other treatment options, including bacteriophage (phage) therapy. To this end, there has been a significant effort to identify new Pseudomonas phages. Here, we isolated and characterized a bacteriophage (termed PIP, pili-infecting phage) that infects P. aeruginosa PA14. Examination of the PIP genome revealed that this phage represents a new species in the subclass Queuovirinae. The isolation and characterization of spontaneous PA14 mutants that are resistant to PIP infection revealed Type IV pili as the PIP receptor. Ultimately, this study characterizes a new species of Pseudomonas phage, thus enhancing the known diversity of phages that infect this important pathogen.

Formation of Genetically Determined Resistance against Human Norovirus Infection through Polymorphism of the FUT2 gene: a Review of the Literature

Relevance. Human Noroviruses (HuNoV) are highly contagious pathogens responsible of acute human norovirus infection. HuNoV is the cause of every fifth case of acute non-bacterial gastroenteritis, annually causing about 699 million cases of the disease and more than 200 thousand deaths worldwide. Controlled expression of the HBGA antigens by the FUT2 gene causes resistance to human norovirus. Polymorphisms of the FUT family genes contribute to partial or complete immunity to certain genogroups/ genotypes of norovirus.Aims. To characterize the effect of FUT2 gene polymorphisms on susceptibility to HuNoV.Results. Nonsensemutations of G428A in two homologous alleles contribute to the formation of a secretory-negative phenotype (se), which is a factor determining immunity to noroviruses. Some missense-mutations in the nucleotide positions se385,571 form partial resistance against certain genotypes. People with a secretory-negative phenotype are immune to infection by the GII.4 genotype and its genovariants.Conclusions. The expression of HLA antigens by the functionally inactive FUT2 gene plays a key role in the resistance of the human population to HuNoV. Susceptibility to HuNoV largely depends on the prevalence of HBGA phenotypic diversity among ethnic populations around the world. Targeted screening aimed at identifying polymorphisms of the FUT family will allow identifying risk groups more susceptible to HuNoV.

Carcinome hépatocellulaire et virus de l’hépatite C, stratégies diagnostiques et thérapeutiques

AbstractHepatitis C virus (HCV) is a hepatotropic RNA virus capable of establishing acute and chronic infections in humans. In the majority of cases, HCV induces persistent infections leading to chronic hepatitis, associated to various degrees with necrotic-inflammatory activity and hepatic fibrosis. The main complications are cirrhosis and hepatocellular carcinoma (HCC), responsible for almost 270,000 deaths a year world-wide.The development of potent, well-tolerated antiviral therapies and the availability of new virological tools have revolutionized management of hepatitis C infection. In line with the objectives of the World Health Organization (WHO), there is an urgent need to simplify and expand screening and diagnosis of HCV infection, simplify the cascade of care and increase access to antiviral treatment.

Assessing the efficacy of different bead-based assays in capturing hepatitis E virus

Hepatitis E virus (HEV) generally causes acute liver infection in humans and its transmission could be waterborne, foodborne, bloodborne, or zoonotic. To date, there is no standard method for the detection of HEV from food and environmental samples. Herein, we explored the possibility of using magnetic beads for the capture and detection of HEV. For this purpose, we employed Dynabeads M-270 Epoxy magnetic beads, coated with different monoclonal antibodies (mAbs) against HEV capsid protein, and the Nanotrap Microbiome A Particle magnetic beads, which are coated with chemical affinity baits, to capture HEV-3 particles in suspension. Viral RNA was extracted by heat-shock or QIAamp viral RNA kit and subjected to quantification using digital-droplet RT-PCR (ddRT-PCR). We demonstrated that the mAb-coupled Dynabeads and the Nanotrap particles, both were able to successfully capture HEV-3. The latter, however had lower limit of detection (<140gc compared with <1400 gc) and significantly higher extraction efficiency in comparison to the mAb-coupled Dynabeads (41.1% vs 8.8%). We have also observed that viral RNA extraction by heat-shock is less efficient compared to using highly denaturing reagents in QIAmp viral RNA extraction kit. As such, magnetic beads have the potential to be used to capture HEV virions for research and surveillance purposes.

Modern view on the features of the development and course of the antiviral immune response

The review article summarizes recent literature data on immunopathogenetic features that influence the nature of the development and course of viral infections.&#x0D; According to the impact on a cell, cytopathic and non-cytopathic viruses are isolated. The course of viral infections is accompanied by cell death, immunopathology, immunosuppression, oncogenesis, and in later stages molecular mimicry and immune amnesia. The acute course of infections is controlled mainly by the mechanisms of innate immunity. The severity of the course of such infections is associated with the genetic variability of viruses and resistance to the antibodies' neutralizing effects. Viruses that are non-cytopathic in their natural mouse hosts can cause acute and chronic infections in humans. The activity of viral reproduction, the persistent infection may be associated with the peculiarities of the expression of gene profiles of the infected cell. The synthesis of endogenous interferons can also affect the nature of the infection development. Disturbances in signaling through Toll-like receptors may contribute to the persistence of infection. Some of the viral proteins block the presentation by the molecules of the main histocompatibility complex of I, II class of viral antigens, interfering with various stages of the presentation. This process is facilitated by a decrease in the transcription of genes of the main histocompatibility complex, blocking the secretion of immunogenic peptides by the proteasome or interfering with the subsequent assembly and transport of the peptide complex to the cell surface. A number of viral proteins stimulate the virus reproduction and inhibit apoptosis. It is believed that the B7-2 molecule is most important for triggering the immune response. Immunodominant epitopes of viral antigens, mutants of cytotoxic lymphocytes are key factors in the immunopathogenesis of persistent, latent infections. Changes in the viral genome of even a single amino acid allow them to avoid recognizing epitopes by an activated T-lymphocyte. Another mechanism for viruses to escape the immune system control is the death of activated T-cells.

Genetic characterization and phylogenetic analysis of human norovirus infection in individual municipalities of the Sverdlovsk region in 2022

Introduction. Human Noroviruses (HuNoV) are highly contagious pathogens responsible of acute human norovirus infection (HuNoVI). GII.4 is the prevailing norovirus genotype in the incidence of gastroenteritis worldwide. Currently there are no studies on the molecular monitoring and phylogenetic analysis of HuNoV in the territory of the Sverdlovsk region, therefore it is not possible to objectively assess their genetic diversity.&#x0D; The aim of the study is to carry out genotyping and phylogenetic analysis of HNoVI in municipalities of the Sverdlovsk region: Ekaterinburg, Nizhny Tagil, Kamensk-Uralsky in 2022.&#x0D; Materials and methods. Fecal samples were collected from children suffering from HuNoVI in the municipalities of the Sverdlovsk region. Viral RNA was extracted from stool samples followed by reverse transcription polymerase chain reaction with degenerate primers flanking the ORF1/ORF2 junction. The amplified samples were genotyped by Sanger sequencing. Multiple sequence alignments were carried out and phylogenetic trees were constructed.&#x0D; Results. The total number of studied samples was 220. According to sequencing results, 73 positive sequences of HuNoV were typed, which represents 33% of the total volume of the studied samples. The largest share of genotypes is occupied by noroviruses GII (58%), noroviruses GI (42%). Noroviruses GII.17, GI.3 were the dominant genotypes. Phylogenetic analysis demonstrates that the identified sequences on the territory of the Sverdlovsk region have the smallest genetic distance, which gives grounds for their unification into a common cluster.&#x0D; Conclusion. For the first time, a genetic analysis of HuNoV was carried out in the territory of the Sverdlovsk region. The norovirus genotyping system based on the amplification of the ORF1/ORF2 region makes it possible to successfully identify various genotypes of noroviruses from the Sverdlovsk region. It has been confirmed that GII noroviruses remain the dominant genetic group. The results of phylogenetic analysis demonstrate the greatest homology in the formed clusters between isolates from Ekaterinburg, Nizhny Tagil, Kamensk-Uralsky.

First serological and molecular investigation of hepatitis E virus infection in dromedary camels in Algeria.

Hepatitis E is an acute self-limited or fulminant infection in humans, caused by the hepatitis E virus (HEV). This member of the Hepeviridae family has been identified in a wide range of domestic and wild animals all over the world, with a possible transmission to humans through fecal oral route, direct contact and ingestion of contaminated meat products, making it one of the global zoonotic and public health major concerns. Since there is no monitoring program and a lack of data on HEV in animals in Algeria, the current preliminary survey has been undertaken to elucidate the exposure to the virus in camels at abattoirs of six southern provinces of Algeria. Two-hundred and eight sera/plasma were collected and analyzed (by double antigen sandwich ELISA) for the presence of total anti-HEV antibodies, among which 35.1% were positive, but no HEV RNA could be isolated from them (by two pan-HEV nested RT-PCR and broad range real-time reverse transcription RT-PCR). The univariate analysis showed significant associations (p < 0.05) between HEV seroprevalence and province of origin, age, and sex of camels, whereas the multivariable logistic regression analysis revealed a negative impact of camels' age on it. The obtained results confirm that HEV infection is widespread established in the camelid population of Algeria.

Unveiling the stealthy tactics: mycoplasma's immune evasion strategies.

Mycoplasmas, the smallest known self-replicating organisms, possess a simple structure, lack a cell wall, and have limited metabolic pathways. They are responsible for causing acute or chronic infections in humans and animals, with a significant number of species exhibiting pathogenicity. Although the innate and adaptive immune responses can effectively combat this pathogen, mycoplasmas are capable of persisting in the host, indicating that the immune system fails to eliminate them completely. Recent studies have shed light on the intricate and sophisticated defense mechanisms developed by mycoplasmas during their long-term co-evolution with the host. These evasion strategies encompass various tactics, including invasion, biofilm formation, and modulation of immune responses, such as inhibition of immune cell activity, suppression of immune cell function, and resistance against immune molecules. Additionally, antigen variation and molecular mimicry are also crucial immune evasion strategies. This review comprehensively summarizes the evasion mechanisms employed by mycoplasmas, providing valuable insights into the pathogenesis of mycoplasma infections.

Pseudomonas aeruginosa biofilm formation and its resistance to beta-lactam antibiotics

ABSTRACT Introduction: An opportunistic pathogen, Pseudomonas aeruginosa is a Gram-negative bacteria that causes acute and chronic human infections. By adhering to appropriate surfaces and creating a biofilm matrix, Pseudomonas aeruginosa has a high-resistance structure. Due to biofilm resistance mechanisms, this bacterial biofilm may increase natural antibiotic resistance. Antibiotics have difficulty penetrating the exopolysaccharide matrix that makes up the biofilm structure (Psl, Pe, alginate, and eDNA). Alginate is involved in generating mucus, and Psl and Pel components are implicated in biofilm development and antibiotic resistance. Due to the connection between biofilm production and antibiotic resistance, Pseudomonas aeruginosa involves mechanisms of beta-lactam antibiotic resistance. Objective: On this basis, learning more about the connection between Pseudomonas aeruginosa bacteria's ability to form biofilms and their resistance to beta-lactam antibiotics is essential. Methods: We use literature methods from various literature on the biofilm formation of P. aeruginosa and its resistance to beta-lactam antibiotics, including research papers and studies with no restrictions types of studies included in this article. Discussion: Pseudomonas aeruginosa has intrinsic and adaptive antibiotic resistance mechanisms. Decreased membrane permeability, the production of enzymes resistant to antibiotics, chromosomal changes, and horizontal gene transfer from other bacteria contribute to antimicrobial resistance. Beta-lactam antibiotic resistance mechanisms are known to be developed by Pseudomonas aeruginosa. The resistance characteristics of Pseudomonas aeruginosa may change phenotypically due to biofilm development. This article will be helpful in future research on Pseudomonas aeruginosa therapy and medication.

Pola Hubungan Faktor Lingkungan Dengan Kejadian Chikungunya Di Wilayah Temanggung Jawa Tengah

In the world around 17% of human infectious diseases are caused by viruses transmitted anthropods such as chikungunya fever. Chikungunya fever is a disease caused by the Chikungunya virus (CHIKV), a type of ribonucleic acid virus (RNA) is transmitted through Aedes aegipty bites. Acute Chikungunya infection in humans can cause sudden fever, joint pain, rashes. Indonesian was reportedly 241 cases of Chikungunya cases in 2021, in the Temanggung Regency until 2022 there were no cases of Chikungunya. On July 26, 2022 there was a suspected Chikungunya in Temanggung I Village as many as 17 people Temanggung Health Center Work Area. The aims of study was to determine relationship between environmental factors and suspected chikungunya. Research used the quantitative with a case control design. The sampling technique uses a total sampling of 17 cases with a sample ratio of 1: 1 (17 cases and 17 controls), analyzed using the Chi-square test. The results showed that environmental factors variables presence of larvae (OR 5,958, p = 0.016), presence water reservoirs (or 4,643, p = 0.037), presence of waste (OR 4,400, p = 0.039), and occupancy density (OR 5,760, p = 0.016) has a significant pattern of relationship with the suspected Chikungunya.

Vemurafenib Inhibits Acute and Chronic Enterovirus Infection by Affecting Cellular Kinase Phosphatidylinositol 4-Kinase Type IIIβ.

Enteroviruses are one of the most abundant viruses causing mild to serious acute infections in humans and also contributing to chronic diseases like type 1 diabetes. Presently, there are no approved antiviral drugs against enteroviruses. Here, we studied the potency of vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, as an antiviral against enteroviruses. We showed that vemurafenib prevented enterovirus translation and replication at low micromolar dosage in an RAF/MEK/ERK-independent manner. Vemurafenib was effective against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect was related to a cellular phosphatidylinositol 4-kinase type IIIβ (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevented infection efficiently in acute cell models, eradicated infection in a chronic cell model, and lowered virus amounts in pancreas and heart in an acute mouse model. Altogether, instead of acting through the RAF/MEK/ERK pathway, vemurafenib affects the cellular PI4KB and, hence, enterovirus replication, opening new possibilities to evaluate further the potential of vemurafenib as a repurposed drug in clinical care. IMPORTANCE Despite the prevalence and medical threat of enteroviruses, presently, there are no antivirals against them. Here, we show that vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, prevents enterovirus translation and replication. Vemurafenib shows efficacy against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect acts through cellular phosphatidylinositol 4-kinase type IIIβ (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevents infection efficiently in acute cell models, eradicates infection in a chronic cell model, and lowers virus amounts in pancreas and heart in an acute mouse model. Our findings open new possibilities to develop drugs against enteroviruses and give hope for repurposing vemurafenib as an antiviral drug against enteroviruses.

Passive Transfer of Animal-Derived Polyclonal Hyperimmune Antibodies Provides Protection of Mice from Lethal Lassa Virus Infection.

Lassa virus (LASV) can cause severe acute systemic infection in humans. No approved antiviral drugs or vaccines are currently available. Antibody-based therapeutics are considered a promising treatment strategy in the management of LASV disease. We used chimeric Ifnar-/- C57BL/6 (Ifnar-/- Bl6) mice, a lethal LASV mouse model, to evaluate the protective efficacy of polyclonal antibodies purified from sera of rabbits hyperimmunized with virus-like particles displaying native-like LASV glycoprotein GP spikes. Polyclonal anti-LASV GP antibodies provided 100% protection against lethal LASV infection in a pre- and post-exposure treatment setting and prevented LASV disease. Treatment also significantly lowered viremia level and virus load in organs. When treatment was initiated at the onset of symptoms, the hyperimmune antibodies provided partial protection and increased the survival rate by 80%. Our findings support the consideration of animal-derived hyperimmune antibodies targeting GP as an effective treatment option for highly pathogenic LASV.

High seroprevalence of hepatitis E virus (HEV) in South Transdanubia, Hungary (2010-2022).

Hepatitis E virus (HEV) is an increasingly recognized etiological agent of acute, chronic and extrahepatic human infections with primarily zoonotic origin in Europe. Limited numbers of comprehensive population-based studies are available related to HEV seroepidemiology, especially from Central Europe.The aim of this study was to investigate the seroprevalence and trends of total and IgM antibodies against HEV in different age groups in the population of South Transdanubia, Hungary, within a thirteen years long period between the years 2010 and 2022.We retrospectively analysed the serological test results of HEV total and HEV IgM antibodies carried out by ELISA technique using Dia.Pro (Diagnostic Bioprobes, Italy) kit from serum samples collected from patients with or without hepatitis between January 1, 2010 and December 31, 2022.The number of tested samples (∑6,996 for total antibody and ∑6,582 for IgM) increased during the study period. The average HEV total and the IgM antibody seropositivities were 33% (2,307/6,996 samples) and 9.6% (642/6,582 samples), respectively, in the study population. The HEV total antibody seropositivity varied in different age groups between 3.9% (age group 1-5 years) and 58.6% (86-90 years) and showed an increasing positivity by age. At the age groups >50 years, nearly half (43%) of the population had antibodies against HEV. The HEV IgM positivity had an increasing trend of up to 13.9% in the age group 81-85 years.High HEV total and IgM antibody seroprevalence were detected in South Transdanubia, Hungary, confirming that this region is highly endemic for HEV infections in Europe.

Identification of Type 4B Secretion System Substrates That Are Conserved among Coxiella burnetii Genomes and Promote Intracellular Growth.

Coxiella burnetii is a Gram-negative pathogen that infects a variety of mammalian hosts. Infection of domesticated ewes can cause fetal abortion, whereas acute human infection normally manifests as the flu-like illness Q fever. Successful host infection requires replication of the pathogen within the lysosomal Coxiella-containing vacuole (CCV). The bacterium encodes a type 4B secretion system (T4BSS) that delivers effector proteins into the host cell. Disruption of C. burnetii T4BSS effector export abrogates CCV biogenesis and bacterial replication. Over 150 C. burnetii T4BSS substrates have been designated often based on heterologous protein translocation by the Legionella pneumophila T4BSS. Cross-genome comparisons predict that many of these T4BSS substrates are truncated or absent in the acute-disease reference strain C. burnetii Nine Mile. This study investigated the function of 32 proteins conserved among diverse C. burnetii genomes that are reported to be T4BSS substrates. Despite being previously designated T4BSS substrates, many of the proteins were not translocated by C. burnetii when expressed fused to the CyaA or BlaM reporter tags. CRISPR interference (CRISPRi) indicated that of the validated C. burnetii T4BSS substrates, CBU0122, CBU1752, CBU1825, and CBU2007 promote C. burnetii replication in THP-1 cells and CCV biogenesis in Vero cells. When expressed in HeLa cells tagged at its C or N terminus with mCherry, CBU0122 localized to the CCV membrane and the mitochondria, respectively. Collectively, these data further define the repertoire of bona fide C. burnetii T4BSS substrates. IMPORTANCE Coxiella burnetii secretes effector proteins via a T4BSS that are required for successful infection. Over 150 C. burnetii proteins are reported to be T4BSS substrates and often by default considered putative effectors, but few have assigned functions. Many C. burnetii proteins were designated T4BSS substrates using heterologous secretion assays in L. pneumophila and/or have coding sequences that are absent or pseudogenized in clinically relevant C. burnetii strains. This study examined 32 previously reported T4BSS substrates that are conserved among C. burnetii genomes. Of the proteins tested that were previously designated T4BSS substrates using L. pneumophila, most were not exported by C. burnetii. Several T4BSS substrates that were validated in C. burnetii also promoted pathogen intracellular replication and one trafficked to late endosomes and the mitochondria in a manner suggestive of effector activity. This study identified several bona fide C. burnetii T4BSS substrates and further refined the methodological criteria for their designation.

PitA Controls the H2- and H3-T6SSs through PhoB in Pseudomonas aeruginosa.

Pseudomonas aeruginosa possesses three type VI secretion systems (T6SSs) that are involved in interspecies competition, internalization into epithelial cells, and virulence. Host-derived mucin glycans regulate the T6SSs through RetS, and attacks from other species activate the H1-T6SS. However, other environmental signals that control the T6SSs remain to be explored. Previously, we determined PitA to be a constitutive phosphate transporter, whose mutation reduces the intracellular phosphate concentration. Here, we demonstrate that mutation in the pitA gene increases the expression of the H2- and H3-T6SS genes and enhances bacterial uptake by A549 cells. We further found that mutation of pitA results in activation of the quorum sensing (QS) systems, which contributes to the upregulation of the H2- and H3-T6SS genes. Overexpression of the phosphate transporter complex genes pstSCAB or knockdown of the phosphate starvation response regulator gene phoB in the ΔpitA mutant reduces the expression of the QS genes and subsequently the H2- and H3-T6SS genes and bacterial internalization. Furthermore, growth of wild-type PA14 in a low-phosphate medium results in upregulation of the QS and H2- and H3-T6SS genes and bacterial internalization compared to those in cells grown in a high-phosphate medium. Deletion of the phoB gene abolished the differences in the expression of the QS and T6SS genes as well as bacterial internalization in the low- and high- phosphate media. Overall, our results elucidate the mechanism of PitA-mediated regulation on the QS system and H2- and H3-T6SSs and reveal a novel pathway that regulates the T6SSs in response to phosphate starvation. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogenic bacterium that causes acute and chronic infections in humans. The type VI secretion systems (T6SSs) have been shown to associate with chronic infections. Understanding the mechanism used by the bacteria to sense environmental signals and regulate virulence factors will provide clues for developing novel effective treatment strategies. Here, we demonstrate a relationship between a phosphate transporter and the T6SSs and reveal a novel regulatory pathway that senses phosphate limitation and controls bacterial virulence factors in P. aeruginosa.

A Library of Promoter-gfp Fusion Reporters for Studying Systematic Expression Pattern of Cyclic-di-GMP Metabolism-Related Genes in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is an environmental microorganism and is a model organism for biofilm research. Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that plays critical roles in biofilm formation. P. aeruginosa contains approximately 40 genes that encode enzymes that participate in the metabolism of c-di-GMP (biosynthesis or degradation), yet it lacks tools that aid investigation of the systematic expression pattern of those genes. In this study, we constructed a promoter-gfp fusion reporter library that consists of 41 reporter plasmids. Each plasmid contains a promoter of corresponding c-di-GMP metabolism-related (CMR) genes from P. aeruginosa reference strain PAO1; thus, each promoter-gfp fusion reporter can be used to detect the promoter activity as well as the transcription of corresponding gene. The promoter activity was tested in P. aeruginosa and Escherichia coli. Among the 41 genes, the promoters of 26 genes showed activity in both P. aeruginosa and E. coli. The library was applied to determine the influence of different temperatures, growth media, and subinhibitory concentrations of antibiotics on the transcriptional profile of the 41 CMR genes in P. aeruginosa. The results showed that different growth conditions did affect the transcription of different genes, while the promoter activity of a few genes was kept at the same level under several different growth conditions. In summary, we provide a promoter-gfp fusion reporter library for systematic monitoring or study of the regulation of CMR genes in P. aeruginosa. In addition, the functional promoters can also be used as a biobrick for synthetic biology studies. IMPORTANCE The opportunistic pathogen P. aeruginosa can cause acute and chronic infections in humans, and it is one of the main pathogens in nosocomial infections. Biofilm formation is one of the most important causes for P. aeruginosa persistence in hosts and evasion of immune and antibiotic attacks. c-di-GMP is a critical second messenger to control biofilm formation. In P. aeruginosa reference strain PAO1, 41 genes are predicted to participate in the making and breaking of this dinucleotide. A major missing piece of information in this field is the systematic expression profile of those genes in response to changing environment. Toward this goal, we constructed a promoter-gfp transcriptional fusion reporter library that consists of 41 reporter plasmids, each of which contains a promoter of corresponding c-di-GMP metabolism-related genes in P. aeruginosa. This library provides a helpful tool to understand the complex regulation network related to c-di-GMP and to discover potential therapeutic targets.

Surveillance, isolation and genomic characterization of Pteropine orthoreovirus of probable bat origin among patients with acute respiratory infection in Malaysia.

Pteropine orthoreovirus (PRV), an emerging bat-borne virus, has been linked to cases of acute respiratory infections (ARI) in humans. The prevalence, epidemiology and genomic diversity of PRV among ARI of unknown origin were studied. Among 632 urban outpatients tested negative for all known respiratory viruses, 2.2% were PRV-positive. Patients mainly presented with moderate to severe forms of cough, sore throat and muscle ache, but rarely with fever. Phylogenetic analysis revealed that over 90% of patients infected with the Melaka virus (MelV)-like PRV, while one patient infected with the Pulau virus previously found only in fruit bats. Human oral keratinocytes and nasopharyngeal epithelial cells were susceptible to clinical isolates of PRV, including the newly isolated MelV-like 12MYKLU1034. Whole genome sequence of 12MYKLU1034 using Nanopore technique revealed a novel reassortant strain. Evolutionary analysis of the global PRV strains suggests the continuous evolution of PRV through genetic reassortment among PRV strains circulating in human, bats and non-human primate hosts, creating a spectrum of reassortant lineages with complex evolutionary characteristics. In summary, the role of PRV as a common etiologic agent of ARI is evident. Continuous monitoring of PRV prevalence, pathogenicity and diversity among human and animal hosts is important to trace the emergence of novel reassortants.