Outer Membrane Protein Research Articles

Proteomic approach to identify host cell attachment proteins provides protective Pseudomonas aeruginosa vaccine antigen FtsZ

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes severe nosocomial infections in susceptible individuals due to the emergence of multidrug-resistant strains. There are no approved vaccines against P. aeruginosa infections nor candidates in active clinical development, highlighting the need for novel candidates and strategies. Using a cell-blot proteomic approach, we reproducibly identified 49 proteins involved in interactions with human lung epithelial cells across four P. aeruginosa strains. Among these were cell division protein FtsZ and outer membrane protein OpmH. Escherichia coli BL21 cells overexpressing recombinant FtsZ or rOpmH showed a 66- and 15-fold increased ability to attach to 16HBE14o− cells, further supporting their involvement in host cell attachment. Both antigens led to proliferation of NK and CD8+ cytotoxic T cells, significant increases in the production of IFN-γ, IL-17A, TNF and IL-4 in immunised mice and elicited strong antigen-specific serological IgG1 and IgG2c responses. Immunisation with FtsZ significantly reduced bacterial burden in the lungs by 1.9-log CFU and dissemination to spleen by 1.8-log CFU. The protective antigen candidate, FtsZ, would not have been identified by traditional approaches relying on either virulence mechanisms or sequence-based predictions, opening new avenues in the development of an anti-P. aeruginosa vaccine.

Ro5-4864, a translocator protein ligand, regulates T cell-mediated inflammatory responses in skin.

Translocator protein (TSPO) is a mitochondrial outer membrane protein expressed on a variety of immune cells, including macrophages, dendritic cells, and T cells, in addition to neurons and steroid-producing cells. Previous studies of TSPO ligands have suggested that TSPO is involved in multiple cellular functions, including steroidogenesis, immunomodulation, and cell proliferation. Currently, there are limited reports on the effects of TSPO or TSPO ligands on T cell-mediated immune responses. We here investigated the involvement of TSPO/TSPO ligand in T cell responses using a 2,4-dinitro-1-fluorobenzene (DNFB)-induced contact hypersensitivity (CH) model. Treatment with Ro5-4864, a TSPO ligand, during DNFB sensitization reduced the number and activation status of CD4+ and CD8+ T cells in draining lymph nodes and alleviated skin inflammation after DNFB challenge. Adoptive transfer of Ro5-4864-treated mouse-derived DNFB-sensitized T cells to naïve mice inhibited CH responses after DNFB challenge. Ro5-4864-treated sensitized T cells showed lower proliferative responses when stimulated with DNFB-pulsed antigen-presenting cells compared to control-treated sensitized T cells. Ro5-4864 also suppressed cell proliferation, as well as adenosine triphosphate and lactate production, during T cell activation. Moreover, the inhibitory effects of Ro5-4864 on T cell responses were conserved in TSPO-deficient cells. Our results suggest that Ro5-4864 inhibits CH responses by suppressing energy metabolism, at least via glycolysis, to reduce the T cell primary response in a TSPO-independent manner.

Outer membrane protein C is a protective and unique vaccine antigen against Shigella flexneri 3a

The anti-Shigella vaccine is one of the WHO’s top priorities. Every year the disease kills more than 200,000 people worldwide and poses a serious threat to children under 5 years of age and the elderly. Increasing antibiotic resistance and limitations in diagnostics emphasize the need to develop an effective vaccine. Recent research and clinical trials report multiple approaches used in Shigella-vaccine development. However, despite the efforts of researchers, pharmaceutical companies and health care organizations, there is no licensed vaccine against shigellosis available to the community. Here, we expressed, broadly characterized and demonstrated the protective properties of outer membrane protein C as an effective molecule serving as a universal antigen for Shigella vaccine. Most of the current approaches to the development of Shigella vaccine are based on the polysaccharide antigens, which are serotype specific and have always been challenging in terms of their high specificity, targeting the most exposed surface antigens identified for certain Shigella serotypes. Here, we confirm immunogenic and protective properties of the recombinant OmpC protein, which protects mice against a lethal dose of a virulent strain 2 weeks after active immunization.

Mitochondrial Bioenergetics of Functional Wound Closure is Dependent on Macrophage-Keratinocyte Exosomal Crosstalk.

Tissue nanotransfection (TNT)-based fluorescent labeling of cell-specific exosomes has shown that exosomes play a central role in physiological keratinocyte-macrophage (mϕ) crosstalk at the wound-site. Here, we report that during the early phase of wound reepithelialization, macrophage-derived exosomes (Exomϕ), enriched with the outer mitochondrial membrane protein TOMM70, are localized in leading-edge keratinocytes. TOMM70 is a 70 kDa adaptor protein anchored in the mitochondrial outer membrane and plays a critical role in maintaining mitochondrial function and quality. TOMM70 selectively recognizes cytosolic chaperones by its tetratricopeptide repeat (TPR) domain and facilitates the import of preproteins lacking a positively charged mitochondrial targeted sequence. Exosomal packaging of TOMM70 in mϕ was independent of mitochondrial fission. TOMM70-enriched Exomϕ compensated for the hypoxia-induced depletion of epidermal TOMM70, thereby rescuing mitochondrial metabolism in leading-edge keratinocytes. Thus, macrophage-derived TOMM70 is responsible for the glycolytic ATP supply to power keratinocyte migration. Blockade of exosomal uptake from keratinocytes impaired wound closure with the persistence of proinflammatory mϕ in the wound microenvironment, pointing toward a bidirectional crosstalk between these two cell types. The significance of such bidirectional crosstalk was established by the observation that in patients with nonhealing diabetic foot ulcers, TOMM70 is deficient in keratinocytes of wound-edge tissues.

Absence of oncomodulin increases susceptibility to noise-induced outer hair cell death and alters mitochondrial morphology

Cochlear outer hair cells (OHCs) play a fundamental role in the hearing sensitivity and frequency selectivity of mammalian hearing and are especially vulnerable to noise-induced damage. The OHCs depend on Ca2+ homeostasis, which is a balance between Ca2+ influx and extrusion, as well as Ca2+ buffering by proteins and organelles. Alterations in OHC Ca2+ homeostasis is not only an immediate response to noise, but also associated with impaired auditory function. However, there is little known about the contribution of Ca2+ buffering proteins and organelles to the vulnerability of OHCs to noise. In this study, we used a knockout (KO) mouse model where oncomodulin (Ocm), the major Ca2+ binding protein preferentially expressed in OHCs, is deleted. We show that Ocm KO mice were more susceptible to noise induced hearing loss compared to wildtype (WT) mice. Following noise exposure (106 dB SPL, 2 h), Ocm KO mice had higher threshold shifts and increased OHC loss and TUNEL staining, compared to age-matched WT mice. Mitochondrial morphology was significantly altered in Ocm KO OHCs compared to WT OHCs. Before noise exposure, Ocm KO OHCs showed decreased mitochondrial abundance, volume, and branching compared to WT OHCs, as measured by immunocytochemical staining of outer mitochondrial membrane protein, TOM20. Following noise exposure, mitochondrial proteins were barely visible in Ocm KO OHCs. Using a mammalian cell culture model of prolonged cytosolic Ca2+ overload, we show that OCM has protective effects against changes in mitochondrial morphology and apoptosis. These experiments suggest that disruption of Ca2+ buffering leads to an increase in noise vulnerability and mitochondrial-associated changes in OHCs.

Investigation of Bacterial Outer Membrane Proteins and Suggesting Antiadhesive Agents from Guava Plant in Skin Infections

Klebsiella pneumoniae is an opportunistic pathogenic bacterium belonging to Enterobacteriaceae family that owns outer membrane proteins called omp35 and omp36 which are responsible for colonization and adhesion to host tissues such as skin. Twenty pre-diagnosed bacterial isolates of K. pneumoniae of wounds and burn swabs were selected in this study, and the diagnosis was confirmed by VITEK-2 automated system. Whole DNA was isolated then amplified by PCR then gel electrophoresis was performed to detect the prevalence of the genes that encode both of omp35 and omp36 in bacterial isolates. Phylogenetic tree of both studied genes was conducted to investigate the precursor of tested isolates in relation to NCBI data. Virtually, the affinity of two medicinal components of fruit Guava (Psidium guajava L.), which are Quercetin and L-arabinopyranoside, were tested as antiadhesive materials against omp35 and omp35 proteins by molecular docking tools. Results showed that 15 out of 20 skin-infecting K. pneumoniae isolates owned both omp35 and omp36 genes as detected by gel electrophoresis and the phylogenetic trees of both genes illustrated that they were related to surgical and nosocomial swab infection precursors in their origin in NCBI. High affinity of Quercetin was recorded towards omp35 protein and omp36 protein which were (-8.1 and -7.8) respectively, in the same time the affinity of L-arabinopyranoside was (-5.2 and -5.3) towards omp35 and omp36 proteins; resulting in suggesting medicinal ingredients of Guava fruit to prevent adhesion and colonization of K. pneumoniae and other Enterobacteriaceae members which might invade wounded skin by bacterial outer membrane proteins.

Outer membrane protein genes and MDR resistance of Enterobacter cloacae in UTI of bladder cancer patients

Outer membrane protein genes and MDR resistance of Enterobacter cloacae in UTI of bladder cancer patients

Pathogenicity of Citrobacter freundii Causing Mass Mortalities of Macrobrachium rosenbergii and Its Induced Host Immune Response.

Citrobacter freundii is an opportunistic pathogen of freshwater aquatic animals, which severely restricts the sustainable development of the aquaculture industry. In this study, a dominant strain, named FSNM-1, was isolated from the hepatopancreas of diseased Macrobrachium rosenbergii. This strain was identified as C. freundii based on a comprehensive analysis of its morphological, physiological, and biochemical features and molecular identification. Challenge experiments were conducted to assess the pathogenicity of C. freundii to M. rosenbergii. The results showed that the FSNM-1 strain had high virulence to M. rosenbergii with a median lethal dose (LD50) of 1.1 × 106 CFU/mL. Histopathological analysis revealed that C. freundii infection caused different degrees of inflammation in the hepatopancreas, gills, and intestines of M. rosenbergii. The detection of virulence-related genes revealed that the FSNM-1 strain carried colonization factor antigen (cfa1, cfa2), ureases (ureG, ureF, ureD, ureE), and outer membrane protein (ompX), and virulence factor detection showed that the FSNM-1 strain had lecithinase, amylase, lipase, gelatinase, and hemolysin activities but did not produce protease and DNase activities. To investigate the immune response of M. rosenbergii to C. freundii, the expression levels of ALF3, MyD88, SOD, proPO, TRAF6, and TNF immune-related genes were monitored at different points of time in the hepatopancreas, gills, intestines, and hemocytes of M. rosenbergii after infection. The results demonstrated a significant upregulation in the expression levels of the ALF3, MyD88, SOD, proPO, TRAF6, and TNF genes in M. rosenbergii at the early stage of C. freundii infection. This study highlights C. freundii as a major pathogen causing mass mortality in M. rosenbergii and provides valuable insights into its virulence mechanisms and the host's immune response.

Evaluation of the Protective Efficacy of Different Doses of a Chlamydia abortus Subcellular Vaccine in a Pregnant Sheep Challenge Model for Ovine Enzootic Abortion.

Chlamydia abortus causes the disease ovine enzootic abortion, which is one of the most infectious causes of foetal death in small ruminants worldwide. While the disease can be controlled using live and inactivated commercial vaccines, there is scope for improvements in safety for both sheep and human handlers of the vaccines. We have previously reported the development of a new prototype vaccine based on a detergent-extracted outer membrane protein preparation of C. abortus that was determined to be more efficacious and safer than the commercial vaccines when administered in two inoculations three weeks apart. In this new study, we have developed this vaccine further by comparing its efficacy when delivered in one or two (1 × 20 µg and 2 × 10 µg) doses, as well as also comparing the effect of reducing the antigen content of the vaccine by 50% (2 × 5 µg and 1 × 10 µg). All vaccine formulations performed well in comparison to the unvaccinated challenge control group, with no significant differences observed between vaccine groups, demonstrating that the vaccine can be administered as a single inoculation and at a lower dose without compromising efficacy. Future studies should focus on further defining the optimal antigen dose to increase the commercial viability of the vaccine.

An ancient ecospecies of Helicobacter pylori.

Helicobacter pylori disturbs the stomach lining during long-term colonization of its human host, with sequelae including ulcers and gastric cancer1,2. Numerous H. pylori virulence factors have been identified, showing extensive geographic variation1. Here we identify a 'Hardy' ecospecies of H. pylori that shares the ancestry of 'Ubiquitous' H. pylori from the same region in most of the genome but has nearly fixed single-nucleotide polymorphism differences in 100 genes, many of which encode outer membrane proteins and host interaction factors. Most Hardy strains have a second urease, which uses iron as a cofactor rather than nickel3, and two additional copies of the vacuolating cytotoxin VacA. Hardy strains currently have a limited distribution, including in Indigenous populations in Siberia and the Americas and in lineages that have jumped from humans to other mammals. Analysis of polymorphism data implies that Hardy and Ubiquitous coexisted in the stomachs of modern humans since before we left Africa and that both were dispersed around the world by our migrations. Our results also show that highly distinct adaptive strategies can arise and be maintained stably within bacterial populations, even in the presence of continuous genetic exchange between strains.

Immunopeptidomics of Salmonella enterica Serovar Typhimurium-Infected Pig Macrophages Genotyped for Class II Molecules.

Salmonellosis is a zoonotic infection that has a major impact on human health; consuming contaminated pork products is the main source of such infection. Vaccination responses to classic vaccines have been unsatisfactory; that is why peptide subunit-based vaccines represent an excellent alternative. Immunopeptidomics was used in this study as a novel approach for identifying antigens coupled to major histocompatibility complex class II molecules. Three homozygous individuals having three different haplotypes (Lr-0.23, Lr-0.12, and Lr-0.21) were thus selected as donors; peripheral blood macrophages were then obtained and stimulated with Salmonella typhimurium (MOI 1:40). Although similarities were observed regarding peptide length distribution, elution patterns varied between individuals; in total, 1990 unique peptides were identified as follows: 372 for Pig 1 (Lr-0.23), 438 for Pig 2 (Lr.0.12) and 1180 for Pig 3 (Lr.0.21). Thirty-one S. typhimurium unique peptides were identified; most of the identified peptides belonged to outer membrane protein A and chaperonin GroEL. Notably, 87% of the identified bacterial peptides were predicted in silico to be elution ligands. These results encourage further in vivo studies to assess the immunogenicity of the identified peptides, as well as their usefulness as possible protective vaccine candidates.

The outer membrane protein, OMP71, of Riemerella anatipestifer, mediates adhesion and virulence by binding to CD46 in ducks

The Riemerella anatipestifer bacterium is known to cause infectious serositis in ducklings. Moreover, its adherence to the host’s respiratory mucosa is a critical step in pathogenesis. Membrane cofactor protein (MCP; CD46) is a complement regulatory factor on the surface of eukaryotic cell membranes. Bacteria have been found to bind to this protein on host cells. Outer membrane proteins (OMPs) are necessary for adhesion, colonisation, and pathogenicity of Gram-negative bacteria; however, the mechanism by which R. anatipestifer adheres to duck cells remains unclear. In this study, pull-down assays and LC–MS/MS identified eleven OMPs interacting with duck CD46 (dCD46), with OMP71 exhibiting the strongest binding. The ability of an omp71 gene deletion strain to bind dCD46 is weaker than that of the wild-type strain, suggesting that this interaction is important. Further evidence of this interaction was obtained by synthesising OMP71 using an Escherichia coli recombinant protein expression system. Adhesion and invasion assays and protein and antibody blocking assays confirmed that OMP71 promoted the R. anatipestifer YM strain (RA-YM) adhesion to duck embryo fibroblasts (DEFs) by binding to CD46. Tests of the pathogenicity of a Δomp71 mutant strain of RA-YM on ducks compared to the wild-type parent supported the hypothesis that OMP71 was a key virulence factor of RA-YM. In summary, the finding that R. anatipestifer exploits CD46 to bind to host cells via OMP71 increases our understanding of the molecular mechanism of R. anatipestifer invasion. The finding suggests potential targets for preventing and treating diseases related to R. anatipestifer infection.

Campycins are novel broad-spectrum antibacterials killing Campylobacter jejuni

Pyocins are high molecular weight bacteriocins produced by Pseudomonas aeruginosa that can be retargeted to new bacterial species by exchanging the pyocin tail fibers with bacteriophage receptor binding proteins (RBPs). Here, we develop retargeted pyocins called campycins as new antibacterials to precisely and effectively kill the major foodborne pathogen Campylobacter jejuni. We used two diverse RBPs (H-fibers) encoded by CJIE1 prophages found in the genomes of C. jejuni strains CAMSA2147 and RM1221 to construct campycin 1 and campycin 2, respectively. Campycins 1 and 2 could target all C. jejuni strains tested due to complementary antibacterial spectra. In addition, both campycins led to more than 3 log reductions in C. jejuni counts under microaerobic conditions at 42 °C, whereas the killing efficiency was less efficient under anaerobic conditions at 5 °C. Furthermore, we discovered that both H-fibers used to construct the campycins bind to the essential major outer membrane protein (MOMP) present in all C. jejuni in a strain-specific manner. Protein sequence alignment and structural modeling suggest that the highly variable extracellular loops of MOMP form the binding sites of the diverse H-fibers. Further in silico analyses of 5000 MOMP sequences indicated that the protein falls into three major clades predicted to be targeted by either campycin 1 or campycin 2. Thus, campycins are promising antibacterials against C. jejuni and are expected to broadly target numerous strains of this human pathogen in nature and agriculture.Key points• Campycins are engineered R-type pyocins containing H-fibers from C. jejuni prophages• Campycins reduce C. jejuni counts by >3 logs at conditions promoting growth• Campycins bind to the essential outer membrane protein MOMP in a strain-dependent way

Cyclic-di-GMP modulates lapA-mediated biofilm formation of Hafnia paralvei to enhance its intestinal colonization and injury

The formation of biofilm by pathogen promotes its colonization, posing significant threats to the environment and the health of humans and animals. The mechanism by which c-di-GMP regulates biofilm formation and its impact on pathogen colonization and intestinal injury has not been fully characterized. Here, we found that overexpressing the phosphodiesterases encoding gene adrB in Hafnia paralvei Z11 reduced c-di-GMP levels, leading to a decrease in outer membrane proteins (OMPs) content and the declining adhesion, as well as a lower level of biofilm formation. Conversely, overexpressing the OMPs-encoding gene lapA significantly enhanced adhesion and biofilm formation. Furthermore, overexpression of adrB exhibited a lower colonization ability in the loach intestine, whereas overexpression of lapA in strain Z11::pBBR1MCS-adrB significantly promoted its colonization in the loach intestine, and caused damage to intestinal barrier integrity, inducing host oxidative stress and the elevated expression level of inflammation-related genes. Thus, this study reveals that c-di-GMP regulates lapA-mediated biofilm formation of H. paralvei Z11, which promotes its intestinal colonization and damage to loach intestines, enhancing our understanding of pathogen-induced intestinal colonization and damage strategies.

Impact of acidic and alkaline conditions on Staphylococcus aureus and Acinetobacter baumannii interactions and their biofilms.

Bacterial biofilms pose significant challenges due to their association with antibiotic resistance, metabolic adaptation, and survival under harsh conditions. Among notable pathogens forming biofilms, Staphylococcus aureus and Acinetobacter baumannii are concerning pathogens in nosocomial settings. However, their behaviour under acidic (pH 4.5) and alkaline (pH10.5) conditions, especially in co-culture setups, remains insufficiently understood. This study investigates these aspects, by examining growth rates, biofilm formation, pH shifts, phenotypic analysis, and gene expression profiles. The results showed A. baumannii exhibited reduced growth and biofilm formation at pH 4.5, while S. aureus showed slow growth and low biofilm formation at pH10.5 in mono-cultures. S. aureus leaned towards an acidic pH (6-6.5), whereas A. baumannii shifted towards an alkaline pH (8-9). In co-culture environments, growth rates and biofilm formation increased across all pH conditions, converging towards a neutral pH over time. Phenotypic motility assays indicated that A. baumannii exhibited greater motility in alkaline conditions, while S. aureus showed increased staphyloxanthin production under acidic conditions. Gene expression analyses revealed that the fibronectin-binding protein A (FnbA) and N-acetylglucosaminyl-transferase (icaA) genes, responsible for initial attachment during biofilm formation, were highly expressed in acidic co-culture condition but poorly expressed in alkaline condition. In A. baumannii, the outer membrane protein A (OmpA) gene associated with adhesion and virulence, was upregulated in co-culture. TheLuxR gene involved in quorum sensing was upregulated in acidic conditions and poorly expressed at pH 10.5. This study elucidates the metabolic adaptability and biofilm formation tendencies of S. aureus towards acidic conditions and A. baumannii towards alkaline conditions, providing insights for better management of biofilm-related infections.

The OprF porin as a potential target for the restoration of carbapenem susceptibility in Pseudomonas aeruginosa expressing acquired carbapenemases.

Carbapenem resistance in Pseudomonas aeruginosa is primarily due to the acquisition of carbapenemases and is often associated with a diminution of the membrane permeability. The outer membrane protein, OprD, is a well-known route, by which carbapenems, predominantly imipenem, can enter the cell, and its loss has been associated with reduced susceptibility to imipenem. In this study, we investigated the antibiotic susceptibility patterns of isogenic P. aeruginosa mutants containing various acquired β-lactamases, including carbapenemases, in a porin-depleted background. We identified that the deletion of oprF was associated with some recovery of susceptibility to carbapenems.

Transcriptome analysis reveals a new virulence-associated trimeric autotransporter responsible for Glaesserella parasuis autoagglutination.

Capsular polysaccharide is an important virulence factor of Glaesserella parasuis. An acapsular mutant displays multiple phenotype variations, while the underlying mechanism for these variations is unknown. In this study, we created an acapsular mutant by deleting the wza gene in the capsule locus. We then used transcriptome analysis to compare the gene expression profiles of the wza deletion mutant with those of the parental strain to understand the possible reasons for the phenotypic differences. The mutant Δwza, which has a deleted wza gene, secreted less polysaccharide and lost its capsule structure. The Δwza exhibited increased autoagglutination, biofilm formation and adherence to eukaryotic cells, while the complementary strain C-Δwza partially restored the phenotype. Transcriptome analysis revealed several differentially expressed genes (DEGs) in Δwza, including up-regulated outer membrane proteins and proteins involved in peptidoglycan biosynthesis, suggesting that wza deletion affects the cell wall homeostasis of G. parasuis. Transcriptome analysis revealed the contribution of non-coding RNAs in the regulation of DEGs. Moreover, a new virulence-associated trimeric autotransporter, VtaA31 is upregulated in Δwza. It is responsible for enhanced autoagglutination but not for enhanced biofilm formation and adherence to eukaryotic cells in Δwza. In conclusion, these data indicate that wza affects the expression of multiple genes, especially those related to cell wall synthesis. Furthermore, they provide evidence that vtaA31 is involved in the autoagglutination of G. parasuis.

A novel method for the preparation of reproducible, stable, and non-infectious quality control materials for Chlamydia trachomatis nucleic acid detection.

Chlamydia trachomatis (CT) is a significant sexually transmitted pathogen known to evoke severe complications, including infertility. Nucleic acid amplification tests (NAATs) are recommended by the World Health Organization to detect CT infection. Furthermore, the establishment of methods, performance validation, internal quality control, and external quality assessment for CT NAATs necessitate the utilization of quality control materials (QCs). QCs are specimens or solutions that are analyzed for quality control purposes in a test system. In this study, we established a novel cell line that stably integrates CT amplification target sequences for producing QCs for CT NAATs. Utilizing clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology, we integrated the CT plasmid-mediated sequence (comprising the full length of the cryptic plasmid and the major outer membrane protein gene, 9,136 bp) into the MUC4 gene of HEK293T cells. Positive clones were screened through flow cytometric sorting, single-cell culture, and PCR-based identification, followed by the establishment of stable cell lines. These cells were then processed using optimized cell preservation procedures to prepare QCs. The sequence insertion copy number was confirmed by real-time quantitative PCR. This novel CT QCs demonstrate excellent clinical applicability, non-infectiousness, quantifiability, and stability. With an integrated sequence exceeding 9 kb in length, it offers exceptional flexibility for adapting to new kit developments. Furthermore, maintaining a well-defined copy number and stable shelf life, the QCs closely aligns with the quality control requirements of CT NAATs. This study presents an innovative method for preparing QCs for CT nucleic acid detection, making a valuable contribution to improving the performance of CT NAATs.IMPORTANCEUntreated CT infections impose significant burdens on individuals and communities, underscoring the importance of early and accurate testing via CT NAATs for disease control. QCs are instrumental in identifying testing process issues. Hence, we developed a cell line integrating CT-amplified target sequences as readily accessible non-infectious QCs. These QCs boast several advantages: the integration of over 9 kb of CT sequence allows for broad applicability, allowing flexible adaptation to the development of new kits. Confirming the CT sequence copy number provides a reliable basis for QC concentration preparation and kit detection limit evaluation. Optimized preservation protocol enhances QC stability during storage, facilitating convenient shipment to clinical laboratories at ambient temperatures. In summary, our novel CT QCs offer a powerful tool for improving CT NAAT performance and present a fresh perspective on QC preparation for detecting nucleic acids from intracellular parasitic pathogens.

First molecular diagnosis of the human pathogen Rickettsia raoultii and other spotted fever group rickettsiae in Sudanese ixodid ticks from domestic ruminants.

Rickettsial infections are often neglected and poorly recognized by physicians in many tropical and subtropical regions. Despite a number of recent reports describing rickettsial diseases in new locations and the discovery of new rickettsiae, medical science and research have largely neglected the diagnosis and antimicrobial treatment of rickettsial infections in subtropical and tropical areas; thus, much remains to be discovered. This study aimed to detect and characterize spotted fever group (SFG) rickettsiae in ixodid ticks infesting domestic ruminants in Khartoum State. Polymerase chain reaction targeting both genes that encode for citrate synthase (gltA) and outer membrane protein (ompA) was performed for the presence of SFG rickettsia followed by sequence and phylogenetic analysis. Of the 202 ticks examined for the presence of SFG rickettsia, gltA gene was detected in 4 samples (2%). Furthermore, gltA-positive samples were used to amplify the ompA gene, in which only two samples yielded positive results. Sequence and phylogenetic analysis of the positive samples revealed four different species of SFG rickettsiae: Rickettsia aeschlimannii, Rickettsia rhipicephali, Rickettsia massiliae and Rickettsia raoultii. These results indicated the presence of SFG rickettsia in Sudanese ticks. This also indicates that humans have an opportunity to acquire these infections. It is important to keep in mind the need for careful consideration of rickettsial infections in individuals with a fever of unknown origin.

The two-component sensor factor envZ influences antibiotic resistance and virulence in the evolutionary dynamics of multidrug-resistant Salmonella enteritidis causing multisite invasive infections.

To assess the impact of mutations in the two-component sensor envZ on antibiotic resistance and virulence in the evolutionary dynamics of MDR Salmonella enteritidis (S. enteritidis). Five S. enteritidis isolates obtained from a patient with multisite invasive infections were analysed. Analysis of antibiotic resistance genes, virulence genes and SNP was performed through WGS. RNA sequencing, quantitative RT-PCR, virulence testing in a Galleria mellonella (G. mellonella) infection model and in vitro cell experiments were used to examine the effects of envZ mutations. WGS revealed identical resistance and virulence genes on an IncFIB(S)/IncFII(S)/IncX1 fusion plasmid in all strains. The faecal strains harboured envZ mutations, reducing outer membrane protein ompD and ompF transcriptional level. Virulence testing demonstrated elevated virulence in envZ mutant strains. In vitro experiments revealed increased adhesion, invasion and phagocytosis resistance in envZ mutants, along with reduced biofilm formation and growth rates. These findings highlight novel genetic locations on envZ influencing antibiotic resistance and virulence in clinical S. enteritidis strains. envZ mutations impact antibiotic resistance by down-regulating ompD and ompF expression and enhance virulence, contributing to multisite infections with increased fitness costs.