A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable Haemophilus influenzae virulence factor.

The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.

Extracellular BSA-degrading SAPs in the rare pathogen Meyerozyma guilliermondii strain SO as potential virulence factors in candidiasis.

Meyerozyma guilliermondii (Candida guilliermondii) is one of the Candida species associated with invasive candidiasis. With the potential for expressing industrially important enzymes, M. guilliermondii strain SO possessed 99 % proteome similarity with the clinical ATCC 6260 isolate and showed pathogenicity towards zebrafish embryos. Recently, three secreted aspartyl proteinases (SAPs) were computationally identified as potential virulence factors in this strain without in vitro verification of SAP activity. The quantification of Candida SAPs activity in liquid broth were also scarcely reported. Thus, this study aimed to characterize M. guilliermondii strain SO's ability to produce SAPs (MgSAPs) in different conditions (morphology and medium) besides analyzing its growth profile. MgSAPs' capability to cleave bovine serum albumin (BSA) was also determined to propose that MgSAPs as the potential virulence factors compared to the avirulent Saccharomyces cerevisiae. M. guilliermondii strain SO produced more SAPs (higher activity) in yeast nitrogen base-BSA-dextrose broth compared to yeast extract-BSA-dextrose broth despite insignificantly different SAP activity in both planktonic and biofilm cells. FeCl3 supplementation significantly increased the specific protein activity (∼40 %). The BSA cleavage by MgSAPs at an acidic pH was proven through semi-quantitative SDS-PAGE, sharing similar profile with HIV-1 retropepsin. The presented work highlighted the MgSAPs on fungal cell wall and extracellular milieu during host infection could be corroborated to the quantitative production in different growth modes presented herein besides shedding lights on the potential usage of retropepsin's inhibitors in treating candidiasis. Molecular and expression analyses of MgSAPs and their deletion should be further explored to attribute their respective virulence effects.

A cyclic lipopeptide from Fusarium graminearum targets plant membranes to promote virulence.

Microbial plant pathogens deploy amphipathic cyclic lipopeptides to reduce surface tension in their environment. While plants can detect these molecules to activate cellular stress responses, the role of these lipopeptides or associated host responses in pathogenesis are not fully clear. The gramillin cyclic lipopeptide is produced by the Fusarium graminearum fungus and is a virulence factor and toxin in maize. Here, we show that gramillin promotes virulence and necrosis in both monocots and dicots by disrupting ion balance across membranes. Gramillin is a cation-conducting ionophore and causes plasma membrane depolarization. This disruption triggers cellular signaling, including a burst of reactive oxygen species (ROS), transcriptional reprogramming, and callose production. Gramillin-induced ROS depends on expression of host ILK1 and RBOHD genes, which promote fungal induction of virulence genes during infection and host susceptibility. We conclude that gramillin's ionophore activity targets plant membranes to coordinate attack by the F. graminearum fungus.

Genomic Characterization of Multidrug-Resistant Enterobacteriaceae Clinical Isolates from Southern Thailand Hospitals: Unraveling Antimicrobial Resistance and Virulence Mechanisms.

The emergence and spread of antimicrobial resistance (AMR) among Enterobacteriaceae pose significant threats to global public health. In this study, we conducted a short-term surveillance effort in Southern Thailand hospitals to characterize the genomic diversity, AMR profiles, and virulence factors of Enterobacteriaceae strains. We identified 241 carbapenem-resistant Enterobacteriaceae, of which 12 were selected for whole-genome sequencing (WGS) and genome analysis. The strains included Proteus mirabilis, Serratia nevei, Klebsiella variicola, Klebsiella aerogenes, Klebsiella indica, Klebsiella grimontii, Phytobacter ursingii, Phytobacter palmae, Kosakonia spp., and Citrobacter freundii. The strains exhibited high levels of multidrug resistance, including resistance to carbapenem antibiotics. Whole-genome sequencing revealed a diverse array of antimicrobial resistance genes (ARGs), with strains carrying genes for ß-lactamase, efflux pumps, and resistance to other antibiotic classes. Additionally, stress response, metal tolerance, and virulence-associated genes were identified, highlighting the adaptability and pathogenic potential of these strains. A plasmid analysis identified several plasmid replicons, including IncA/C2, IncFIB(K), and Col440I, as well as several plasmids identical to those found globally, indicating the potential for the horizontal gene transfer of ARGs. Importantly, this study also identified a novel species of Kosakonia spp. PSU27, adding to the understanding of the genetic diversity and resistance mechanisms of Enterobacteriaceae in Southern Thailand. The results reported in this study highlight the critical importance of implementing effective antimicrobial management programs and developing innovative treatment approaches to urgently tackle AMR.

Removal of intracellular and extracellular antibiotic resistance genes and virulence factor genes using electricity-intensified constructed wetlands.

Constructed wetland (CW) is considered a promising technology for the removal of emerging contaminants. However, its removal performance for antibiotic resistance genes (ARGs) is not efficient and influence of virulence factor genes (VFGs) have not been elucidated. Here, removal of intracellular and extracellular ARGs as well as VFGs by electricity-intensified CWs was comprehensively evaluated. The two electrolysis-intensified CWs can improve the removal of intracellular ARGs and MGEs to 0.96- and 0.85-logs, respectively. But cell-free extracellular ARGs (CF-eARGs) were significantly enriched with 1.8-logs in the electrolysis-intensified CW. Interestingly, adding Fe-C microelectrolysis to the electrolysis-intensified CW is conducive to the reduction of CF-eARGs. However, the detected number and relative abundances of intracellular and extracellular VFGs were increased in all of the three CWs. The biofilms attached onto the substrates and rhizosphere are also hotspots of both intracellular and particle-associated extracellular ARGs and VFGs. Structural equation models and correlation analysis indicated that ARGs and VFGs were significantly cooccurred, suggesting that VFGs may affect the dynamics of ARGs. The phenotypes of VFGs, such as biofilm, may act as protective matrix for ARGs, hindering the removal of resistance genes. Our results provide novel insights into the ecological remediation technologies to enhance the removal of ARGs.

adhesiomeR: a tool for Escherichia coli adhesin classification and analysis.

Adhesins are crucial factors in the virulence of bacterial pathogens such as Escherichia coli. However, to date no resources have been dedicated to the detailed analysis of E. coli adhesins. Here, we provide adhesiomeR software that enables characterization of the complete adhesin repertoire, termed the adhesiome. AdhesiomeR incorporates the most comprehensive database of E. coli adhesins and facilitates an extensive analysis of adhesiome. We demonstrate that adhesiomeR achieves 98% accuracy when compared with experimental analyses. Based on analysis of 15,000 E. coli genomes, we define novel adhesiome profiles and clusters, providing a nomenclature for a unified comparison of E. coli adhesiomes.

Structural and functional insights into the C-terminal signal domain of the Bacteroidetes type-IX secretion system.

Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 ß-sandwich (ß1-ß7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus ('motif C-t.') and the loop connecting strands ß3 and ß4 ('motif Lß3ß4') as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS, which shares the same general topology as in Porphyromonas CTDs. However, motif Lß3ß4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.

Identification of a putative α-galactoside ß-(1 → 3)-galactosyltransferase involved in the biosynthesis of galactomannan side chain of glucuronoxylomannogalactan in Cryptococcus neoformans.

The cell surface of Cryptococcus neoformans is covered by a thick capsular polysaccharide. The capsule is the most important virulence factor of C. neoformans; however, the complete mechanism of its biosynthesis is unknown. The capsule is composed of glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal). As GXM is the most abundant component of the capsule, many studies have focused on GXM biosynthesis. However, although GXMGal has an important role in virulence, studies on its biosynthesis are scarce. Herein, we have identified a GT31 family ß-(1 → 3)-galactosyltransferase Ggt2, which is involved in the biosynthesis of the galactomannan side chain of GXMGal. Comparative analysis of GXMGal produced by a ggt2 disruption strain revealed that Ggt2 is a glycosyltransferase that catalyzes the initial reaction in the synthesis of the galactomannan side chain of GXMGal. The ggt2 disruption strain showed a temperature-sensitive phenotype at 37°C, indicating that the galactomannan side chain of GXMGal is important for high-temperature stress tolerance in C. neoformans. Our findings provide insights into complex capsule biosynthesis in C. neoformans.

Catabolite control protein C contributes to virulence and hydrogen peroxide-induced oxidative stress responses in Listeria monocytogenes.

Listeria monocytogenes causes listeriosis, an infectious and potentially fatal disease of animals and humans. A diverse network of transcriptional regulators, including LysR-type catabolite control protein C (CcpC), is critical for the survival of L. monocytogenes and its ability to transition into the host environment. In this study, we explored the physiological and genetic consequences of deleting ccpC and the effects of such deletion on the ability of L. monocytogenes to cause disease. We found that ccpC deletion did not impact hemolytic activity, whereas it resulted in significant reductions in phospholipase activities. Western blotting revealed that the ΔccpC strain produced significantly reduced levels of the cholesterol-dependent cytolysin LLO relative to the wildtype F2365 strain. However, the ΔccpC mutant displayed no significant intracellular growth defect in macrophages. Furthermore, ΔccpC strain exhibited reduction in plaque numbers in fibroblasts compared to F2365, but plaque size was not significantly affected by ccpC deletion. In a murine model system, the ΔccpC strain exhibited a significantly reduced bacterial burden in the liver and spleen compared to the wildtype F2365 strain. Interestingly, the deletion of this gene also enhanced the survival of L. monocytogenes under conditions of H2O2-induced oxidative stress. Transcriptomic analyses performed under H2O2-induced oxidative stress conditions revealed that DNA repair, cellular responses to DNA damage and stress, metalloregulatory proteins, and genes involved in the biosynthesis of peptidoglycan and teichoic acids were significantly induced in the ccpC deletion strain relative to F2365. In contrast, genes encoding internalin, 1-phosphatidylinositol phosphodiesterase, and genes associated with sugar-specific phosphotransferase system components, porphyrin, branched-chain amino acids, and pentose phosphate pathway were significantly downregulated in the ccpC deletion strain relative to F2365. This finding highlights CcpC as a key factor that regulates L. monocytogenes physiology and responses to oxidative stress by controlling the expression of important metabolic pathways.

Protein function annotation and virulence factor identification of Klebsiella pneumoniae genome by multiple machine learning models.

Klebsiella pneumoniae is a type of Gram-negative bacterium which can cause a range of infections in human. In recent years, an increasing number of strains of K. pneumoniae resistant to multiple antibiotics have emerged, posing a significant threat to public health. The protein function of this bacterium is not well known, thus a systematic investigation of K. pneumoniae proteome is in urgent need. In this study, the protein functions of this bacteria were re-annotated, and their function groups were analyzed. Moreover, three machine learning models were built to identify novel virulence factors. Results showed that the functions of 16 uncharacterized proteins were first annotated by sequence alignment. In addition, K. pneumoniae proteins share a high proportion of homology with Haemophilus influenzae and a low homology proportion with Chlamydia pneumoniae. By sequence analysis, 10 proteins were identified as potential drug targets for this bacterium. Our model achieved a high accuracy of 0.901 in the benchmark dataset. By applying our models to K. pneumoniae, we identified 39 virulence factors in this pathogen. Our findings could provide novel clues for the treatment of K. pneumoniae infection.

Nocardia implantans sp. nov. isolated from a patient with pulmonary infection.

TwoGram-stain-positive and rod-shaped actinomycetes (strains CDC186T and CDC192) were isolated from sputum samples of a patient in Chongqing, PR China, and were investigated to determine their taxonomic status. The results of phylogenetic analysis based on the 16S rRNA gene indicated that CDC186T and CDC192 represented members of the genus Nocardia, and the sequence similarity with Nocardia beijingensis DSM 44636T was the highest, at 99.71 and 99.78 %, respectively. The DNA G+C content of both CDC186T and CDC192 was 69.1 %. Genomic diversity analysis revealed that the average nucleotide identity and in silico DNA‒DNA hybridisation values between the two novel strains and closely related species were significantly below the thresholds of 95-96 and 70 %, respectively, but these values between the two novel strains were 99.96 and 99.90 %, respectively. The phylogenetic relationship based on the dapb1 gene and the single-copy core genes further indicated that the two novel strains were clustered in separate branch adjacent to N. beijingensis DSM 44636T. Growth occurred within the ranges of 20-42 °C, pH 6.0-9.0 and NaCl concentrations of 0.5-4.5 % (w/v). The major fatty acids of CDC186T and CDC192 were C16 : 0 and C18 : 0 10-methyl [tuberculostearic acid (TBSA)]. The predominant respiratory menaquinone was MK-9. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside, one unidentified glycolipid, one unidentified phospholipid and one unidentified phosphoglycolipid. All the genomes of the studied strains were annotated with virulence factor (VF)-associated genes homologous to those of Mycobacterium tuberculosis, and the results of susceptibility testing indicated that CDC186T and CDC192 were resistant to amoxicillin-clavulanic acid and tigecycline. On the basis of chemotaxonomic characteristics and the results of phylogenetic analyses, strains CDC186T and CDC192 represent a novel species within the genus Nocardia, for which the name Nocardia implantans sp. nov. is proposed. The type strain is CDC186T (=GDMCC 4.206T= JCM 34959T).

Genome-wide patterns of noncoding and protein-coding sequence variation in the major fungal pathogen Aspergillus fumigatus.

Aspergillus fumigatus is a deadly fungal pathogen, responsible for >400,000 infections/year and high mortality rates. A. fumigatus strains exhibit variation in infection-relevant traits, including in their virulence. However, most A. fumigatus protein-coding genes, including those that modulate its virulence, are shared between A. fumigatus strains and closely related nonpathogenic relatives. We hypothesized that A. fumigatus genes exhibit substantial genetic variation in the noncoding regions immediately upstream to the start codons of genes, which could reflect differences in gene regulation between strains. To begin testing this hypothesis, we identified 5,812 single-copy orthologs across the genomes of 263 A. fumigatus strains. In general, A. fumigatus noncoding regions showed higher levels of sequence variation compared with their corresponding protein-coding regions. Focusing on 2,482 genes whose protein-coding sequence identity scores ranged between 75 and 99%, we identified 478 total genes with signatures of positive selection only in their noncoding regions and 65 total genes with signatures only in their protein-coding regions. Twenty-eight of the 478 noncoding regions and 5 of the 65 protein-coding regions under selection are associated with genes known to modulate A. fumigatus virulence. Noncoding region variation between A. fumigatus strains included single-nucleotide polymorphisms and insertions or deletions of at least a few nucleotides. These results show that noncoding regions of A. fumigatus genes harbor greater sequence variation than protein-coding regions, raising the hypothesis that this variation may contribute to A. fumigatus phenotypic heterogeneity.

The presence of virulence factor genes downregulates uterine AQP3 and alters glutathione peroxidase activity and uterine histopathology in canine pyometra.

Present study was designed to evaluate the role of virulence factor genes (papG, cnf1 and hylA) in the pathogenesis of canine pyometra. Antimicrobial susceptibility test and detection of virulence genes were performed Escherichia coli (E. coli) detected in uterine swab samples. Animals were divided into two groups based on the presence (VF+, n:14) or absence (VF-, n:7) of the virulence factor genes papG, cnf1 and hylA. Blood and tissue glutathione peroxidase activity, uterine histopathologic analysis and AQP3, ESR1, PGR, OXTR gene expressions were determined in both groups. Statistical analyses were performed using Stata version 15.1. All E. coli isolates were susceptible to amikacin, whereas resistant to ampicillin, amoxicillin/clavulanic acid and lincomycin. None of the isolates were susceptible to cefotaxime. E. coli isolates had at least one virulence gene. The most prevalent gene was fimH (100%), followed by fyuA (95.8%), usp (83.3%), sfa (75%), cnf1 and hlyA (70.8%) genes. Blood GPx activity was greater in VF+ animals. On the other hand, uterine tissue GPx activity was lower in VF+ group compared to the control group. Expression levels of AQP3 were upregulated more than fivefold in VF-dogs compared to the control group. In addition, AQP3 expression levels were found approximately threefold higher in VF (-) than VF (+) group (p < .05). Varying degree of inflammation noted for all animals with pyometra, but the presence of bacteria noted only in VF+ animals. In conclusion, the presence of virulence factor genes does not play a role in the histopathological degree of inflammation, the presence of bacteria was found to vary. Serum GPx activity increased in VF+ animals. While the hormone receptor expressions were similar, AQP expression was upregulated in the absence of virulence factor genes.

Leishmania donovani mitogen-activated protein kinases as a host-parasite interaction interface.

Leishmaniasis, a major globally re-emerging neglected tropical disease, has a restricted repertoire of chemotherapeutic options due to a narrow therapeutic index, drug resistance, or patient non-compliance due to toxicity. The disease is caused by the parasite Leishmania that resides in two different forms in two different environments: as sessile intracellular amastigotes within mammalian macrophages and as motile promastigotes in sandfly gut. As mitogen-activated protein kinases (MAPKs) play important roles in cellular differentiation and survival, we studied the expression of Leishmania donovani MAPKs (LdMAPKs). The homology studies by multiple sequence alignment show that excepting LdMAPK1 and LdMAPK2, all thirteen other LdMAPKs share homology with human ERK and p38 isoforms. Expression of LdMAPK4 and LdMAPK5 is less in avirulent promastigotes and amastigotes. Compared to miltefosine-sensitive L. donovani parasites, miltefosine-resistant parasites have higher LdMAPK1, LdMAPK3-5, LdMAPK7-11, LdMAPK13, and LdMAPK14 expression. IL-4-treatment of macrophages down-regulated LdMAPK11, in virulent amastigotes whereas up-regulated LdMAPK5, but down-regulated LdMAPK6, LdMAPK12-15, expression in avirulent amastigotes. IL-4 up-regulated LdMAPK1 expression in both virulent and avirulent amastigotes. IFN-γ-treatment down-regulated LdMAPK6, LdMAPK13, and LdMAPK15 in avirulent amastigotes but up-regulated in virulent amastigotes. This complex profile of LdMAPKs expression among virulent and avirulent parasites, drug-resistant parasites, and in amastigotes within IL-4 or IFN-γ-treated macrophages suggests that LdMAPKs are differentially controlled at the host-parasite interface regulating parasite survival and differentiation, and in the course of IL-4 or IFN-γ dominated immune response.

C5a peptidase (ScpA) activity towards human type II and type III interferons.

C5a peptidase, also known as ScpA, is a surface associated serine protease derived from Streptococcus pyogenes and has been described as an important factor in streptococcus virulence, capable of cleaving complement components C5a, C3 and C3a. Although the interactions of ScpA with complement components is well studied, extensive screening of ScpA activity against other pro-inflammatory cytokines is lacking. Here, ScpA's ability to cleave human pro-inflammatory cytokines was tested, revealing its ability to cleave human IFNγ, IFNλ1, IFNλ2, C5, IL-37 but with significantly reduced activities. The functional consequence of ScpA's cleavage of IFNγ in its signalling through the Jak-Stat pathway has also been evaluated in an in vitro RPE1 cell model. These newly identified targets for ScpA highlight the complexity of streptococcus infections and indeed, the potential for ScpA to have a therapeutic role in the progression of inflammatory diseases involving these cytokines.

Facultative symbiont virulence determines horizontal transmission rate without host specificity in Dictyostelium discoideum social amoebas.

In facultative symbioses, only a fraction of hosts are associated with symbionts. Specific host and symbiont pairings may be the result of host-symbiont coevolution driven by reciprocal selection or priority effects pertaining to which potential symbiont is associated with a host first. Distinguishing between these possibilities is important for understanding the evolutionary forces that affect facultative symbioses. We used the social amoeba, Dictyostelium discoideum, and its symbiont, Paraburkholderia bonniea, to determine whether ongoing coevolution affects which host-symbiont strain pairs naturally cooccur within a facultative symbiosis. Relative to other Paraburkholderia, including another symbiont of D. discoideum, P. bonniea features a reduced genome size that indicates a significant history of coevolution with its host. We hypothesized that ongoing host-symbiont coevolution would lead to higher fitness for naturally cooccurring (native) host and symbiont pairings compared to novel pairings. We show for the first time that P. bonniea symbionts can horizontally transmit to new amoeba hosts when hosts aggregate together during the social stage of their life cycle. Here we find evidence for a virulence-transmission trade-off without host specificity. Although symbiont strains were significantly variable in virulence and horizontal transmission rate, hosts and symbionts responded similarly to associations in native and novel pairings. We go on to identify candidate virulence factors in the genomes of P. bonniea strains that may contribute to variation in virulence. We conclude that ongoing coevolution is unlikely for D. discoideum and P. bonniea. The system instead appears to represent a stable facultative symbiosis in which naturally cooccurring P. bonniea host and symbiont pairings are the result of priority effects.

Prevalence, antimicrobial resistance and detection of virulence genes of Escherichia coli and Salmonella spp. isolated from white-lipped peccaries and collared peccaries.

Salmonella spp. and Escherichia coli are implicated in human and animal infections and require antimicrobial treatment in many situations. Faecal samples of healthy white-lipped peccaries (Pecari tajacu) (n = 30) and collared peccaries (Tayassu pecari ) (n = 60) obtained in three farms located in the Midwest Brazil. The antimicrobial profiles of commensal E. coli from P. tajacu and T. pecari from commercial herds in Brazil were isolated and analyzed and virulence genes were detected. Among 90 healthy animals, no Salmonella spp. were isolated. However, 30 samples (27%) tested positive for E. coli, with 18 isolates from P. tajacu and 12 from T. pecari, representing frequencies of 58.0% and 38.7%, respectively. Additionally, other Enterobacteriaceae family bacteria were detected but not included in this analysis. However, individual samples from 30 animals tested positive for E. coli, of which 16 were isolated from P. tajacu presenting multidrug resistance and six were isolated from T. pecari presenting a similar pattern. The E. coli virulence genes detected were papC (pilus-associated pyelonephritis) in five isolates, tsh (temperature-sensitive hemagglutinin) in one isolate, and eae (enteric attachment and effacement) in one isolate. The serum resistance gene, iss (increased serum survival), was detected in four isolates. An association between these genes and the presence of hemolysin was also observed in one isolate. Thus, T. pecari and P. tajacu are potential reservoirs of pathogenic and multidrug-resistant and E. coli. Faecal E. coli of healthy P. tajacu and T. pecari could act as a possible reservoir of antimicrobial resistance genes in environment.

Direct α-Hydroxy Acid Loading onto a Bacterial Thiotemplate Assembly Line via a Multienzyme Gateway.

Various nonribosomal peptide synthetases (NRPSs) create structural and functional diversity by incorporating α-hydroxy acids into peptide backbones. Trigonic acid, an unusual cyclopropanol-substituted hydroxy acid, is the source of the molecular warhead of malleicyprol, a critical virulence factor of human and animal pathogens of the Burkholderia pseudomallei (BP) group. The process of selecting and loading this building block remained enigmatic as the NRPS module designated for this task is incomplete. Using a combination of bioinformatics, mutational analyses, targeted metabolomics, and in vitro biochemical assays, we show that two trans-acting enzymes are required to load this central building block onto the modular assembly line. An adenylation-thiolation didomain enzyme (BurJ) activates trigonic acid, followed by the translocation of the enzyme-bound α-hydroxy acid thioester by an FkbH-like protein with a mutated phosphatase domain (BurH). This specialized gateway is the first reported direct loading of an α-hydroxy acid onto a bona fide NRPS module in bacteria and expands the synthetic biology toolbox for the site-specific incorporation of non-canonical building blocks. Moreover, insight into the biochemical basis of virulence factor biosynthesis can provide a foundation for developing enzyme inhibitors as anti-virulence therapeutics against BP pathogen infections.

Comprehensive genomic landscape of antibiotic resistance in Staphylococcus epidermidis.

Staphylococcus epidermidis, a common commensal bacterium found on human skin, can cause infections in clinical settings, and the presence of antibiotic resistance genes (ARGs) impedes the treatment of S. epidermidis infections. However, studies characterizing the ARGs in S. epidermidis with regard to genomic and ecological diversities are limited. Thus, we performed a comprehensive and comparative analysis of 405 high-quality S. epidermidis genomes, including those of 35 environmental isolates from the Han River, to investigate the genomic diversity of antibiotic resistance in this pathogen. Comparative genomic analysis revealed the prevalence of ARGs in S. epidermidis genomes associated with multi-locus sequence types. The genes encoding dihydrofolate reductase (dfrC) and multidrug efflux pump (norA) were genome-wide core ARGs. ß-Lactam class ARGs were also highly prevalent in the S. epidermidis genomes, which was consistent with the resistance phenotype observed in river isolates. Furthermore, we identified chloramphenicol acetyltransferase genes (cat) in the plasmid-like sequences of the six river isolates, which have not been reported previously in S. epidermidis genomes. These genes were identical to those harbored by the Enterococcus faecium plasmids and associated with the insertion sequence 6 family transposases, homologous to those found in Staphylococcus aureus plasmids, suggesting the possibility of horizontal gene transfer between these Gram-positive pathogens. Comparison of the ARG and virulence factor profiles between S. epidermidis and S. aureus genomes revealed that these two species were clearly distinguished, suggesting genomic demarcation despite ecological overlap. Our findings provide a comprehensive understanding of the genomic diversity of antibiotic resistance in S. epidermidis. IMPORTANCE: A comprehensive understanding of the antibiotic resistance gene (ARG) profiles of the skin commensal bacterium and opportunistic pathogen Staphylococcus epidermidis needs to be documented from a genomic point of view. Our study encompasses a comparative analysis of entire S. epidermidis genomes from various habitats, including those of 35 environmental isolates from the Han River sequenced in this study. Our results shed light on the distribution and diversity of ARGs within different S. epidermidis multi-locus sequence types, providing valuable insights into the ecological and genetic factors associated with antibiotic resistance. A comparison between S. epidermidis and Staphylococcus aureus revealed marked differences in ARG and virulence factor profiles, despite their overlapping ecological niches.

Multi-omics strategy reveals potential role of antimicrobial resistance and virulence factor genes responsible for Simmental diarrheic calves caused by Escherichia coli.

Escherichia coli (E. coli) is reported to be an important pathogen associated with calf diarrhea. Antibiotic resistance genes (ARGs) and virulence factor genes (VFGs) pose a considerable threat to both animal and human health. However, little is known about the characterization of ARGs and VFGs presented in the gut microbiota of diarrheic calves caused by E. coli. In this study, we used multi-omics strategy to analyze the ARG and VFG profiles of Simmental calves with diarrhea caused by E. coli K99. We found that gut bacterial composition and their microbiome metabolic functions varied greatly in diarrheic calves compared to healthy calves. In total, 175 ARGs were identified, and diarrheal calves showed a significantly higher diversity and abundance of ARGs than healthy calves. Simmental calves with diarrhea showed higher association of VFGs with pili function, curli assembly, and ferrienterobactin transport of E. coli. Co-occurrence patterns based on Pearson correlation analysis revealed that E. coli had a highly significant (P < 0.0001) correlation coefficient (>0.8) with 16 ARGs and 7 VFGs. Metabolomics analysis showed that differentially expressed metabolites in Simmental calves with diarrhea displayed a high correlation with the aforementioned ARGs and VFGs. Phylotype analysis of E. coli genomes showed that the predominant phylogroup B1 in diarrheic Simmental calves was associated with 10 ARGs and 3 VFGs. These findings provide an overview of the diversity and abundance of the gut microbiota in diarrheic calves caused by E. coli and pave the way for further studies on the mechanisms of antibiotic resistance and virulence in the calves affected with diarrhea.IMPORTANCESimmental is a well-recognized beef cattle breed worldwide. They also suffer significant economic losses due to diarrhea. In this study, fecal metagenomic analysis was applied to characterize the antibiotic resistance gene (ARG) and virulence factor gene (VFG) profiles of diarrheic Simmental calves. We identified key ARGs and VFGs correlated with Escherichia coli isolated from Simmental calves. Additionally, metabolomics analysis showed that differentially expressed metabolites in Simmental calves with diarrhea displayed a high correlation with the aforementioned ARGs and VFGs. Our findings provide an insight into the diversity and abundance of the gut microbiota in diarrheic calves caused by Escherichia coli and pave the way for further studies on the mechanisms of antibiotic resistance and virulence in the diarrheal calves from cattle hosts.