Major Virulence Factor Research Articles

The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function.

This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

Vaccine efficacy induced by virus-like particles containing Leishmania donovani surface glycoprotein GP63.

Leishmania donovani surface glycoprotein 63 (GP63) is a major virulence factor involved in parasite escape and immune evasion. In this study, we generated virus-like particles (VLPs) expressing L. donovani GP63 using the baculovirus expression system. Mice were intramuscularly immunized with GP63-VLPs and challenged with L. donovani promastigotes. GP63-VLP immunization elicited higher levels of L. donovani antigen-specific serum antibodies and enhanced splenic B cell, germinal center B cell, CD4+, and CD8+ T cell responses compared to unimmunized controls. GP63-VLPs inhibited the influx of pro-inflammatory cytokines IFN-γ and IL-6 in the livers, as well as thwarting the development of splenomegaly in immunized mice. Upon L. donovani challenge infection, a drastic reduction in splenic parasite burden was observed in VLP-immunized mice. These results indicate that GP63-VLPs immunization conferred protection against L. donovani challenge infection by inducing humoral and cellular immunity in mice.

The chaperone PrsA2 regulates the secretion, stability, and folding of listeriolysin O during Listeria monocytogenes infection.

Lm is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, Lm relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. Lm encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major Lm virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that Lm and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.

RIL-seq reveals extensive involvement of small RNAs in virulence and capsule regulation in hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) can infect healthy individuals, in contrast to classical strains that commonly cause nosocomial infections. The recent convergence of hypervirulence with carbapenem-resistance in K. pneumoniae can potentially create 'superbugs' that are challenging to treat. Understanding virulence regulation of hvKp is thus critical. Accumulating evidence suggest that posttranscriptional regulation by small RNAs (sRNAs) plays a role in bacterial virulence, but it has hardly been studied in K. pneumoniae. We applied RIL-seq to a prototypical clinical isolate of hvKp to unravel the Hfq-dependent RNA-RNA interaction (RRI) network. The RRI network is dominated by sRNAs, including predicted novel sRNAs, three of which we validated experimentally. We constructed a stringent subnetwork composed of RRIs that involve at least one hvKp virulence-associated gene and identified the capsule gene loci as a hub target where multiple sRNAs interact. We found that the sRNA OmrB suppressed both capsule production and hypermucoviscosity when overexpressed. Furthermore, OmrB base-pairs within kvrA coding region and partially suppresses translation of the capsule regulator KvrA. This agrees with current understanding of capsule as a major virulence and fitness factor. It emphasizes the intricate regulatory control of bacterial phenotypes by sRNAs, particularly of genes critical to bacterial physiology and virulence.

Challenges of Diphtheria Toxin Detection.

Diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis. Moreover, new Corynebacterium species with the potential to produce diphtheria toxin have also been described. Therefore, the detection of the toxin is the most important test in the microbiological diagnosis of diphtheria and other corynebacteria infections. Since the first demonstration in 1888 that DT is a major virulence factor of C. diphtheriae, responsible for the systemic manifestation of the disease, various methods for DT detection have been developed, but the diagnostic usefulness of most of them has not been confirmed on a sufficiently large group of samples. Despite substantial progress in the science and diagnostics of infectious diseases, the Elek test is still the basic recommended diagnostic test for DT detection. The challenge here is the poor availability of an antitoxin and declining experience even in reference laboratories due to the low prevalence of diphtheria in developed countries. However, recent and very promising assays have been developed with the potential for use as rapid point-of-care testing (POCT), such as ICS and LFIA for toxin detection, LAMP for tox gene detection, and biosensors for both.

The role and function mechanism of tapP in modulating the virulence of Aeromonas hydrophila

Aeromonas hydrophila (A. hydrophila) is a widespread pathogen capable of infecting most freshwater fish and causing septicemia. Type IV pili are one of the major virulence factors of A. hydrophila and play a key role in virulence and adhesion to the host. TapP is known to be involved in the synthesis of type IV pili. However, the effect and molecular mechanism of the tapP in A. hydrophila remain unknown. In this study, we constructed a genetically stable tapP deletion mutant strain of A. hydrophila (ΔtapP-AH), the median lethal dose (LD50) value of the ΔtapP-AH in Carassius auratus gibelio was 3.1-fold higher than that of the wild-type strain (WT-AH). The transmission electron microscopy (TEM) results showed that ΔtapP-AH exhibited sparser pili. The ability of adhering to gill tissues and biofilm formation was significantly reduced in ΔtapP-AH. For motility, the swimming ability of ΔtapP-AH was significantly higher, no significant difference was found in twitching and swarming abilities. We further investigated the effect of tapP gene deletion on the expression of virulence related factor genes to explore the molecular mechanisms of attenuated virulence and altered biological characteristics. The results indicated that deletion of the tapP directly affected the expression of genes related to type IV pili, type II secretion system, type VI secretion system and flagellum, but had no effect on the genes related to type I pili. In conclusion, this study provides new insights into the function of the tapP and the pathogenic mechanism in A. hydrophila.

MOLECULAR IDENTIFICATION AND GENOTYPING OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) IN DIFFERENT CLINICAL SAMPLES

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious human pathogen that can spread in healthcare facilities and among the general public. This study was aimed to evaluate the prevalence and diversity of SCCmec types of this superbug among hospitalized patients. This study involved phenotypic identification and molecular confirmation of S. aureus based on the nuc gene, molecular detection of MRSA, SCCmec typing, and virulence factor profiling of MRSA clinical isolates obtained from hospitalized patients in Duhok province. Out of the 310 enrolled patients, 33 isolates (10.64%) were identified and confirmed as Staphylococcus aureus, of which 51.5% were identified as MRSA based on phenotypic and molecular targeting of the mecA gene. There were no discernible variations between the prevalence rates of this pathogen in different clinical sources, sexes, or age groups (p-values: 0.71, 0.39, and 0.15 respectively). The isolates had elevated rates of resistance to most antibiotic classes. They were classified as extensive drug-resistant (30.3%), multidrug-resistant (57.5%), and non-multidrug-resistant (12.1%). Additionally, SCCmec typing of MRSA by multiplex PCR identified three different SCCmec types and subtypes, including SCCmec type II (35.5%), followed by 17.64% of SCCmec type IV subtype d (IVd), and SCCmec type III (11.76%). However, 35.3% of the MRSA isolates were found to be non-typeable. Molecular profiling of major virulence factors and toxin genes revealed that 57.5% of the isolates were positive for the exfolitative toxin (ETA), 45.4% of the isolates carried TSST-1 (Toxic Shock Syndrome Toxin-1), the PVL (Panton-Valentine Leukocidin) cytotoxin was identified in 15% of the isolates, and 18.1% of the identified S. aureus isolates were positive for the ACME (arginine catabolic mobile element). The findings of the current investigation pointed out the circulating of highly virulent and extensively resistant MRSA strains among hospitalized patients, which may require active surveillance and better control policies

The interplay between electron transport chain function and iron regulatory factors influences melanin formation in Cryptococcus neoformans.

Mitochondrial functions are critical for the ability of the fungal pathogen Cryptococcus neoformans to cause disease. However, mechanistic connections between key functions such as the mitochondrial electron transport chain (ETC) and virulence factor elaboration have yet to be thoroughly characterized. Here, we observed that inhibition of ETC complex III suppressed melanin formation, a major virulence factor. This inhibition was partially overcome by defects in Cir1 or HapX, two transcription factors that regulate iron acquisition and use. In this regard, loss of Cir1 derepresses the expression of laccase genes as a potential mechanism to restore melanin, while HapX may condition melanin formation by controlling oxidative stress. We hypothesize that ETC dysfunction alters redox homeostasis to influence melanin formation. Consistent with this idea, inhibition of growth by hydrogen peroxide was exacerbated in the presence of the melanin substrate L-DOPA. In addition, loss of the mitochondrial chaperone Mrj1, which influences the activity of ETC complex III and reduces ROS accumulation, also partially overcame antimycin A inhibition of melanin. The phenotypic impact of mitochondrial dysfunction was consistent with RNA-Seq analyses of WT cells treated with antimycin A or L-DOPA, or cells lacking Cir1 that revealed influences on transcripts encoding mitochondrial functions (e.g., ETC components and proteins for Fe-S cluster assembly). Overall, these findings reveal mitochondria-nuclear communication via ROS and iron regulators to control virulence factor production in C. neoformans.IMPORTANCEThere is a growing appreciation of the importance of mitochondrial functions and iron homeostasis in the ability of fungal pathogens to sense the vertebrate host environment and cause disease. Many mitochondrial functions such as heme and iron-sulfur cluster biosynthesis, and the electron transport chain (ETC), are dependent on iron. Connections between factors that regulate iron homeostasis and mitochondrial activities are known in model yeasts and are emerging for fungal pathogens. In this study, we identified connections between iron regulatory transcription factors (e.g., Cir1 and HapX) and the activity of complex III of the ETC that influence the formation of melanin, a key virulence factor in the pathogenic fungus Cryptococcus neoformans. This fungus causes meningoencephalitis in immunocompromised people and is a major threat to the HIV/AIDS population. Thus, understanding how mitochondrial functions influence virulence may support new therapeutic approaches to combat diseases caused by C. neoformans and other fungi.

Tailored multivalent peptide targeting the B-subunit pentamer of cholera toxin inhibits its intestinal toxicity by inducing aberrant transport of the toxin in cells

Cholera toxin (Ctx) is a major virulence factor produced by Vibrio cholerae that can cause gastrointestinal diseases, including severe watery diarrhea and dehydration, in humans. Ctx binds to target cells through multivalent interactions between its B-subunit pentamer and the receptor ganglioside GM1 present on the cell surface. Here, we identified a series of tetravalent peptides that specifically bind to the receptor-binding region of the B-subunit pentamer using affinity-based screening of multivalent random-peptide libraries. These tetravalent peptides efficiently inhibited not only the cell-elongation phenotype but also the elevated cAMP levels, both of which are induced by Ctx treatment in CHO cells or a human colon carcinoma cell line (Caco-2 cells), respectively. Importantly, one of these peptides, NRR-tet, which was highly efficient in these two activities, markedly inhibited fluid accumulation in the mouse ileum caused by the direct injection of Ctx. In consistent, NRR-tet reduced the extensive Ctx-induced damage of the intestinal villi. After NRR-tet bound to Ctx, the complex was incorporated into the cultured epithelial cells and accumulated in the recycling endosome, affecting the retrograde transport of Ctx from the endosome to the Golgi, which is an essential process for Ctx to exert its toxicity in cells. Thus, NRR-tet may be a novel type of therapeutic agent against cholera, which induces the aberrant transport of Ctx in the intestinal epithelial cells, detoxifying the toxin.

An ex vivo Approach in European Seabass Leucocytes Supports the in vitro Regulation by Postbiotics of Aip56 Gene Expression of Photobacterium damselae subsp. piscicida.

Shewanella putrefaciens Pdp11 (SpPdp11) is a probiotic strain assayed in aquaculture; however, its postbiotic potential is unknown. Postbiotics are bacterial metabolites, including extracellular products (ECPs) that improve host physiology and immunity. Their production and composition can be affected by different factors such as the growing conditions of the probiotics. Photobacterium damselae subsp. piscicida strain Lg 41/01 (Phdp) is one of the most important pathogens in marine aquaculture. The major virulent factor of this bacterium is the exotoxin aip56, responsible for inducing apoptosis of fish leucocytes. Viable SpPdp11 cells have been reported to increase resistance to challenges with Phdp. This work aimed to evaluate the effect of two ECPs, T2348-ECP and FM1548-ECP, obtained from SpPdp11 grown under different culture conditions that previously demonstrated to exert different degradative and non-cytotoxic activities, as well as the effect on pathogens biofilm formation. These SpPdp11-ECPs were then analyzed by their effect on the viability, phagocytosis, respiratory burst and apoptogenic activity against European sea bass leucocytes infected or not with Phdp supernatant. Both ECPs, T2348-ECP and FM1548-ECP, were not cytotoxic against leucocytes and significantly reduced their apoptosis. Phagocytosis and respiratory burst of leucocytes were significantly reduced by incubation with Phdp supernatant, and not influenced by incubation with T2348-ECP or FM1548-ECP. However, both activities were significantly increased after leucocyte incubation with combined T2348-ECP and FM1548-ECP with Phdp supernatant, compared to those incubated only with Phdp supernatant. Finally, both T2348-ECP and FM1548-ECP significantly reduced the relative in vitro expression of the Phdp aip56 encoding gene.

Virulence Genes and Biofilm Formation Among Legionella pneumophila Isolates Collected from Hospital Water Sources.

Legionella pneumophila can be transmitted to people, especially immunocompromised patients, via hospital water pipe systems and cause severe pneumonia. The aim of our study was to investigate the presence of major virulence factor genes, ability of biofilms formation, and correlation between presence of Legionella isolates and temperature, pH, and residual chlorine of water. Hundred water samples were collected from nine hospitals in Tehran, Iran. Temperature, pH, and residual chlorine were determined during sampling. Different virulence genes and the ability to form biofilms were subsequently analyzed among the L. pneumophila isolates. Results showed that 12 (12%) samples were positive in culture method and all of the isolates were positive as L. pneumophila species (mip). A correlation was found between Legionella culture positivity and temperature and pH of water, but there was no significant correlation between residual chlorine of water samples and the presence of Legionella. The isolation of Legionella rate in summer and spring was higher than winter and autumn. Twelve (100%) isolates were positive for mip genes, 9 (75%) for dot genes, 8 (66.66%) for hsp, 6 (50%) for lvh, and 4 (33.33%) for rtx. All of the isolates displayed strong ability for biofilm production every three days. Two of these isolates (16.6%) displayed weak ability to form biofilm on the first day of incubation. This study revealed that water sources in hospitals were colonized by virulent Legionella and should be continuously monitored to avoid elevated concentrations of Legionella with visible biofilm formation.

HtpG-A Major Virulence Factor and a Promising Vaccine Antigen against Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading global cause of death f rom an infectious bacterial agent. Therefore, limiting its epidemic spread is a pressing global health priority. The chaperone-like protein HtpG of M. tuberculosis (Mtb) is a large dimeric and multi-domain protein with a key role in Mtb pathogenesis and promising antigenic properties. This dual role, likely associated with the ability of Heat Shock proteins to act both intra- and extra-cellularly, makes HtpG highly exploitable both for drug and vaccine development. This review aims to gather the latest updates in HtpG structure and biological function, with HtpG operating in conjunction with a large number of chaperone molecules of Mtb. Altogether, these molecules help Mtb recovery after exposure to host-like stress by assisting the whole path of protein folding rescue, from the solubilisation of aggregated proteins to their refolding. Also, we highlight the role of structural biology in the development of safer and more effective subunit antigens. The larger availability of structural information on Mtb antigens and a better understanding of the host immune response to TB infection will aid the acceleration of TB vaccine development.

Molecular Detection, Antibiotic Resistance, and Biofilm Formation of Clinical Pseudomonas aeruginosa Isolates from Patients in Al-Hillah, Babylon Province, Iraq

Abstract Background: Pseudomonas aeruginosa is known as one of the main causes of nosocomial infections and is repeatedly related to opportunistic infections among hospitalized patients worldwide. Antibiotic resistance (AR) and biofilm-forming ability (BFA) are two major virulence factors of P. aeruginosa isolates that are involved in the continuation of infections. Objectives: This study aimed to isolate and identify P. aeruginosa isolates from patients and to elucidate the correlation between AR and BFA among isolates. Materials and Methods: A total of 130 clinical P. aeruginosa isolates were obtained from Al-Hillah Al-Sadiq Hospital in Babylon province, Iraq during the period from March to July 2022. AR profile and BFA of P. aeruginosa isolates were proceeded by Kirby-Bauer and Congo red agar (CRA) methods, respectively. Polymerase chain reaction (PCR) screening, targeting the ecfX gene, was conducted to identify species-specific ecfX genes in P. aeruginosa. Results: Multidrug-resistant (MDR) P. aeruginosa accounted for 96.1% (125/130) and 85.4% (111/130) of isolates showed BFA. Statistical analyses showed a nonsignificant correlation between AR and BFA in P. aeruginosa isolates. The ecfX PCR product analyses affirmed their allocation to P. aeruginosa isolates. Conclusion: The high incidence of MDR and BFA among P. aeruginosa isolates might require a plan for regular evaluation and controlling policy programs.

SARS-CoV2 Nsp1 is a metal-dependent DNA and RNA endonuclease.

Over recent years, we have been living under a pandemic, caused by the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). One of the major virulence factors of Coronaviruses is the Non-structural protein 1 (Nsp1), known to suppress the host cells protein translation machinery, allowing the virus to produce its own proteins, propagate and invade new cells. To unveil the molecular mechanisms of SARS-CoV2 Nsp1, we have addressed its biochemical and biophysical properties in the presence of calcium, magnesium and manganese. Our findings indicate that the protein in solution is a monomer and binds to both manganese and calcium, with high affinity. Surprisingly, our results show that SARS-CoV2 Nsp1 alone displays metal-dependent endonucleolytic activity towards both RNA and DNA, regardless of the presence of host ribosome. These results show Nsp1 as new nuclease within the coronavirus family. Furthermore, the Nsp1 double variant R124A/K125A presents no nuclease activity for RNA, although it retains activity for DNA, suggesting distinct binding sites for DNA and RNA. Thus, we present for the first time, evidence that the activities of Nsp1 are modulated by the presence of different metals, which are proposed to play an important role during viral infection. This research contributes significantly to our understanding of the mechanisms of action of Coronaviruses.

The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of this pathogen is type III secretion system 2 (T3SS2), which is encoded on the 80-kb V. parahaemolyticus pathogenicity island (Vp-PAI), the gene expression of which is governed by the OmpR-family transcriptional regulator VtrB. Here, we found a positive autoregulatory feature of vtrB transcription, which is often observed with transcriptional regulators of bacteria, but the regulation was not canonically dependent on its own promoter. Instead, this autoactivation was induced by heterogeneous transcripts derived from the VtrB-regulated operon upstream of vtrB. VtrB-activated transcription overcame the intrinsic terminator downstream of the operon, resulting in transcription read-through with read-in transcription of the vtrB gene and thus completing the autoregulatory loop for vtrB gene expression. The dampening of read-through transcription with an exogenous strong terminator reduced vtrB gene expression. Furthermore, a V. parahaemolyticus mutant with defects in the vtrB autoregulatory loop also showed compromises in T3SS2 expression and T3SS2-dependent cytotoxicity in vitro and enterotoxicity in vivo, indicating that this autoregulatory loop is essential for sustained vtrB activation and the consequent robust expression of T3SS2 genes for pathogenicity. Taken together, these findings demonstrate that the regulatory loop for vtrB gene expression based on read-through transcription from the upstream operon is a crucial pathway in T3SS2 gene regulatory network to ensure T3SS2-mediated virulence of V. parahaemolyticus.

In vitro and in silico assessment of anti-biofilm and anti-quorum sensing properties of 2,4-Di-tert butylphenol against Acinetobacter baumannii.

Introduction. Acinetobacter baumannii is a nosocomial pathogen with a high potential to cause food-borne infections. It is designated as a critical pathogen by the World Health Organization due to its multi-drug resistance and mortalities reported. Biofilm governs major virulence factors, which promotes drug resistance in A. baumannii. Thus, a compound with minimum selection pressure on the pathogen can be helpful to breach biofilm-related virulence.Hypothesis/Gap Statement. To identify anti-biofilm and anti-virulent metabolites from extracts of wild Mangifera indica (mango) brine pickle bacteria that diminishes pathogenesis and resistance of A. baumannii.Aim. This study reports anti-biofilm and anti-quorum sensing (QS) efficacy of secondary metabolites from bacterial isolates of fermented food origin.Method. Cell-free supernatants (CFS) of 13 bacterial isolates from fermented mango brine pickles were screened for their efficiency in inhibiting biofilm formation and GC-MS was used to identify its metabolites. Anti-biofilm metabolite was tested on early and mature biofilms, pellicle formation, extra polymeric substances (EPS), cellular adherence, motility and resistance of A. baumannii. Gene expression and in silico studies were also carried out to validate the compounds efficacy.Results. CFS of TMP6b identified as Bacillus vallismortis, inhibited biofilm production (83.02 %). Of these, major compound was identified as 2,4-Di-tert-butyl phenol (2,4-DBP). At sub-lethal concentrations, 2,4-DBP disrupted both early and mature biofilm formation. Treatment with 2,4-DBP destructed in situ biofilm formed on glass and plastic. In addition, key virulence traits like pellicle (77.5 %), surfactant (95.3 %), EPS production (3-fold) and cell adherence (65.55 %) reduced significantly. A. baumannii cells treated with 2,4-DBP showed enhanced sensitivity towards antibiotics, oxide radicals and blood cells. Expression of biofilm-concomitant virulence genes like csuA/B, pgaC, pgaA, bap, bfmR, katE and ompA along with QS genes abaI, abaR significantly decreased. The in silico studies further validated the higher binding affinity of 2,4-DBP to the AbaR protein than the cognate ligand molecule.Conclusion. To our knowledge, this is the first report to demonstrate 2,4- DBP has anti-pathogenic potential alone and with antibiotics by in vitro, and in silico studies against A. baumannii. It also indicates its potential use in therapeutics and bio-preservatives.

Oxidative stress is intrinsic to staphylococcal adaptation to fatty acid synthesis antibiotics

Antibiotics inhibiting the fatty acid synthesis pathway (FASII) of the major pathogen Staphylococcus aureus reach their enzyme targets, but bacteria continue growth by using environmental fatty acids (eFAs) to produce phospholipids. We assessed the consequences and effectors of FASII-antibiotic (anti-FASII) adaptation. Anti-FASII induced lasting expression changes without genomic rearrangements. Several identified regulators affected the timing of adaptation outgrowth. Adaptation resulted in decreased expression of major virulence factors. Conversely, stress responses were globally increased and adapted bacteria were more resistant to peroxide killing. Importantly, pre-exposure to peroxide led to faster anti-FASII-adaptation by stimulating eFA incorporation. This adaptation differs from reports of peroxide-stimulated antibiotic efflux, which leads to tolerance. Invivo, anti-FASII-adapted S.aureus killed the insect host more slowly but continued multiplying. We conclude that staphylococcal adaptation to FASII antibiotics involves reprogramming, which decreases virulence and increases stress resistance. Peroxide, produced by the host to combat infection, favors anti-FASII adaptation.

Streptococcus agalactiae serotype Ia capsular polysaccharide production from a Brazilian strain

The pathogen S. agalactiae, categorized as Lancefield group B streptococci (GBS), is a Gram-positive bacterium, encapsulated, anaerobic facultative bacillus, which occurs in pairs or short chains. It was responsible for more than 319,000 cases of infections in neonates. Early and late streptococci, in the course of an illness or medical condition, are infections that have high mortality and morbidity rates, with an incidence of 2.0 to 5.0 per 1000 live births in North America, Europe, and Australia. The infection of newborns usually happens through transmission from infected mothers during and after childbirth, with GBS being among the critical bacterial infections in the Brazilian epidemiological picture. S. agalactiae strains isolated from different regions of Brazil, indicate a great diversity among the isolated serotypes but with a higher incidence of serotype Ia. Publications report that, in pregnant women in Brazil, the prevalence of serotype Ia has been constant between 2002 and 2018, followed by serotypes II and IV. Serotypes Ib, III, and V also appear, but with some fluctuations in different regions of the country. The pathogen which causes invasive disease are usually surrounded by a polysaccharide capsule and is its major virulence factor, being that the key antigen in protective response triggered by polysaccharides and, more recently, protein-polysaccharide conjugate vaccines. The development of antibiotic resistance by these microorganisms to different antibiotics has been the guideline in leading therapeutic options against these infections including the use of prophylactic vaccines in pregnant women, immunosuppressed or older people. Such prophylactic alternatives can be constructed from capsular polysaccharides.Based on the experience in the production and purification of Neisseria meningitidis polysaccharides for vaccines, Bio-Manguinhos developed a process for the production, purification and characterization of S. agalactiae Ia polysaccharides. In the present study, the capsular polysaccharide from the strain isolated in clinical material of a public hospital, in Rio de Janeiro – Brazil, was obtained from simple batch cultures, with purification process achieving about 20 mg g−1 in yield for cell dry weight (CDW) and ca. of 100 % of purity. Hence, to prove that the CPS of GBS Ia can be consistently produced, purified, and characterized by NMR, HRGC-MS, and SEC-MALS – from such strain – to be used in vaccine productions against S. agalactiae.

Construction of a Colorimetric and Fluorescence Dual-Mode Immunoassay Detection of Alpha-Hemolysin in Milk.

Alpha-hemolysin (Hla) is a major virulence factor secreted by Staphylococcus aureus (S. aureus), which can lyse a variety of mammalian cells and help bacteria evade the host immune system or antibiotics, posing a safety hazard to human health. Therefore, it is critical to establish a quick-responsive and sensitive method for Hla detection to ensure food safety. In this work, a dual-mode immunoassay was developed with both colorimetric and fluorescent readouts for discriminative detection of Hla. The proposed sensing system consists of p-phenylenediamine (PPD) and fluorescein, where fluorescein functions as a fluorescent reporter, and PPD serves a dual function as a colorimetric reporter and fluorescence quencher. Subsequently, the reaction system of this method was optimized, and the detection limit, sensitivity, and specificity were evaluated. Under optimal conditions, the proposed method possesses excellent analytical performance in the range from 0.5 to 500 ng/mL with a limit of detection as low as 0.5 ng/mL. Noteworthy, this method was successfully employed for the detection of Hla in milk with good selectivity and high accuracy. Overall, the dual-mode immunoassay provides a superior platform for the on-site, quantitative, and accurate detection of Hla in food samples.

تأثير دقائق اوكسيد الزنك النانوية المصنعة بايولوجيًا على النمط الظاهري والجيني للغشاء الحيوي لبكتريا Proteus mirabilis

تصنف بكتريا Proteus mirabilis كثالث مسبب لالتهاب المجاري البولية المصاحب لتركيب أنابيب القسطرة البولية. تعد الاغشية الحيوية من عوامل الضراوة اللتي تستعملها البكتريا لمقاومة المضادات الحيوية وفي ضل تطور مقاومة البكتريا للمضادات الحيوية, صار لزامًا على العلماء والباحيثن في مجال الاحياء المجهرية أيجاد بدائل ذات كفائة عالية قادرة على اختراق جدار الخلية البكتيرية وبالتالي قتلها.في السنوات الاخيرة, اكتسبت دقائق النانو المصنعة بايولوجيا والصديقة للبيئة أهتمامًا كبيرًا من قبل الباحثين لأمتلاكها خصائص مميزة كصغر حجمها المتناهي واشكالها المختلفة يمَكنهم من استعمالها كمواد مضادة للبكتيريا في مختلف المجلات الطبية والصحية. تهدف هذه الدراسة الى التحري عن قدرة دقائق اوكسيد النحاس النانوية المصنعة بايولوجيا على تثبيط تكوين الغشاء الحيوي المنتج من قبل بكتيريا Proteus mirabilis. أنتجت هذه الدقائق بأستعمال راشح بكتيري يحتوي على الانزيمات المختزلة لملح النحاس واستخدمت عدة تقنيات لتحديد الصفات الفيزيائية المظهرية لدقائق النانو المنتجة حيث استخدمت تقنية XRD لتأكيد التركيب خماسي الشكل لهذه الدقائق , بينما استعملت تقنية TEM لتحديد قطر الجزيئات المنتجة بمعدل 84.45 نانومتر. اما FESEM فقد تم استخدامه لتحديد الصفات الخارجية لسطح جزيئات النانو المنتجة , تم تأكيد النتائج بأستخدام تقنية AFM للكشف عن توزيع و وعورة جزيئات النانو.اما UV فقد سجلت ااقصى امتصاصية على الطول الموجي nm287 .تم التحري في هذه الدراسة عن فعالية دقائق اوكسيد النحاس النانوية في منع تكون الغشاء الحيوي حيث أستعمل مستحلب دقائق اوكسيد النحاس النانوي بالتركيز تحت القاتل 32 μg/ml . سجلت نتائج هذه الدراسة انخفاضا ملحوضَا في انتاج الغشاء الحيوي للبكتريا المنتجه له حيث سجلت العزلات ذات الانتاجية العالية للغشاء الحيوي ضعفأ في الانتاج بعد معاملتها بالتركيز المستعمل من دقائق اوكسيد النحاس النانوية. جاء تأثير هذه الدقائق على كل من النمط الظاهري والتعبير الجيني لجين LuxS المسؤول عن تكوين الغشاء الحيوي بأستخدام تقنية rtPCRحيث سجلت الدراسة انخفاضا في التعبير الجيني لهذا الجين بعد معاملة سلالات البكتريا قيد الدراسة بدقائق اوكسيد النحاس النانوية مقارنتاً بالعزلات غير المعاملة. من خلال نتائج هذه التجربة يمكن التوصل الى امكانية اعتماد دقائق اوكسيد الزنك النانوية كعامل مضاد للبكتيريا عن استخدامه بالتركيز المناسب.