Secretion System Research Articles

Iron starvation increases the production of the Pseudomonas aeruginosa RsmY and RsmZ sRNAs in static conditions.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that induces virulence gene expression in response to host-mediated iron starvation. Recently, our laboratory showed that some virulence factors are responsive to iron limitation in static but not shaking growth conditions. One of these is the HSI-2-type six secretion system (T6SS), which is also induced during chronic infection. Iron regulation of T6SS was partially impacted by the iron-responsive PrrF sRNA and completely dependent upon the Pseudomonas quinolone signal (PQS) biosynthetic gene pqsA. Here, we analyzed the impact of iron on the expression of two small regulatory RNAs (sRNAs), RsmY and RsmZ, that activate the expression of T6SS by sequestering the RsmA translation inhibitor. Our results demonstrate that iron starvation induces the expression of RsmY and RsmZ in static but not shaking cultures. We further show that this induction occurs through the rsmY and rsmZ promoters and is dependent upon PqsA. Disruption of either the pqsR gene also eliminated iron-dependent regulation of rsmY and rsmZ promoter activity. Taken together, our results show novel targets of iron regulation that are specific to static growth, highlighting the importance of studying regulatory mechanisms in static communities that may be more representative of growth during chronic infection.IMPORTANCEIron is a central component of various bacterial metabolic pathways making it an important host-acquired nutrient for pathogens to establish infection. Previous iron regulatory studies primarily relied on shaking bacterial cultures; while these ensure cultural homogeneity, they do not reflect growth conditions during infection. We recently showed that static growth of Pseudomonas aeruginosa promotes iron-dependent regulation of a type six secretion system (T6SS), a virulence factor that is induced during chronic infections. In the current study, we found that static growth also promotes iron-dependent regulation of the RsmY and RsmZ sRNAs, which are global regulators that affect T6SS during chronic P. aeruginosa lung infection. Hence, our work demonstrates the Rsm sRNAs as potential effectors of iron regulation during static growth that may also be relevant in chronic infection.

Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

Human genetic associations of the airway microbiome in chronic obstructive pulmonary disease.

Little is known about the relationships between human genetics and the airway microbiome. Deeply sequenced airway metagenomics, by simultaneously characterizing the microbiome and host genetics, provide a unique opportunity to assess the microbiome-host genetic associations. Here we performed a co-profiling of microbiome and host genetics with the identification of over 5 million single nucleotide polymorphisms (SNPs) through deep metagenomic sequencing in sputum of 99 chronic obstructive pulmonary disease (COPD) and 36 healthy individuals. Host genetic variation was the most significant factor associated with the microbiome except for geography and disease status, with its top 5 principal components accounting for 12.11% of the microbiome variability. Within COPD individuals, 113 SNPs mapped to candidate genes reported as genetically associated with COPD exhibited associations with 29 microbial species and 48 functional modules (P < 1 × 10-5), where Streptococcus salivarius exhibits the strongest association to SNP rs6917641 in TBC1D32 (P = 9.54 × 10-8). Integration of concurrent host transcriptomic data identified correlations between the expression of host genes and their genetically-linked microbiome features, including NUDT1, MAD1L1 and Veillonella parvula, TTLL9 and Stenotrophomonas maltophilia, and LTA4H and Haemophilus influenzae. Mendelian randomization analyses revealed a potential causal link between PARK7 expression and microbial type III secretion system, and a genetically-mediated association between COPD and increased relative abundance of airway Streptococcus intermedius. These results suggest a previously underappreciated role of host genetics in shaping the airway microbiome and provide fresh hypotheses for genetic-based host-microbiome interactions in COPD.

Genome Sequence Resource of a Taro Bacterial Soft Rot Pathogen, Dickeya fangzhongdai ZXC1

Dickeya fangzhongdai ZXC1 is a newly identified highly virulent causal agent of taro bacterial soft rot disease isolated from a taro sample showing typical soft rot symptoms in Shaoguan, Guangdong Province, China. The pathogen produces plant cell wall–degrading enzymes that macerate the taro tube tissues. In this study, we report the whole-genome sequencing analysis of strain ZXC1. The results showed that strain ZXC1 has one circular DNA chromosome of 5,129,951 bp with a 56.59% G+C content. In addition to sharing a conserved zms gene cluster that encodes the genes for biosynthesis of phytotoxin zeamines and 11 copies of predicted pectate lyase genes, the strain ZXC1 genome contains more prophage loci and a higher number of type IV secretion system and type IV secretion system gene clusters than the D. fangzhongdai strains isolated from pears or phalaenopsis, which may account for its strong virulence phenotype. The data from this study present a valuable resource for elucidation of the pathogenic mechanisms of D. fangzhongdai and may aid in developing new disease control approaches to safeguard taro production. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Chlamydiae in corals: shared functional potential despite broad taxonomic diversity

Abstract Cnidarians, such as corals and sea anemones, associate with a wide range of bacteria that have essential functions, including nutrient cycling and the production of antimicrobial compounds. Within cnidarians, bacteria can colonize all microhabitats including the tissues. Among them are obligate intracellular bacteria of the phylum Chlamydiota (chlamydiae) whose impact on cnidarian hosts and holobionts, especially corals, remain unknown. Here, we conducted a meta-analysis of previously published 16S rRNA gene metabarcoding data from cnidarians (e.g., coral, jellyfish, anemones), eight metagenome-assembled genomes (MAGs) of coral-associated chlamydiae, and one MAG of jellyfish-associated chlamydiae to decipher their diversity and functional potential. While the metabarcoding dataset showed an enormous diversity of cnidarian-associated chlamydiae, six out of nine MAGs were affiliated with the Simkaniaceae family. The other three MAGs were assigned to the Parasimkaniaceae, Rhabdochlamydiaceae, and Anoxychlamydiaceae, respectively. All MAGs lacked the genes necessary for an independent existence, lacking any nucleotide or vitamin and most amino acid biosynthesis pathways. Hallmark chlamydial genes, such as a type III secretion system, nucleotide transporters, and genes for host interaction, were encoded in all MAGs. Together these observations suggest an obligate intracellular lifestyle of coral-associated chlamydiae. No unique genes were found in coral-associated chlamydiae, suggesting a lack of host specificity. Additional studies are needed to understand how chlamydiae interact with their coral host, and other microbes in coral holobionts. This first study of the diversity and functional potential of coral-associated chlamydiae improves our understanding of both the coral microbiome and the chlamydial lifestyle and host range.

Cohabiting with ulcerative colitis patients decreases differences of gut microbiome between healthy individuals and the patients

Background: Ulcerative colitis (UC), which is characterized by chronic relapsing inflammation of the colon, results from a complex interaction of factors involving the host, environment, and microbiome. The present study aimed to investigate the gut microbial composition and metabolic variations in patients with UC and their spouses. Materials and Methods: Fecal samples were collected from 13 healthy spouses and couples with UC. 16S rRNA gene amplicon sequencing and metagenomics sequencing were used to analyze gut microbiota composition, pathways, gene expression, and enzyme activity, followed by the Kyoto Encyclopedia of Genes and Genomes. Results: We found that the microbiome diversity of couples with UC decreased, especially that of UC patients. Bacterial composition, such as Firmicutes, was altered between UC patients and healthy controls, but was not significantly different between UC patients and their spouses. This has also been observed in pathways, such as metabolism, genetic information processing, organismal systems, and human diseases. However, the genes and enzymes of spouses with UC were not significantly different from those of healthy individuals. Furthermore, the presence of Faecalibacterium correlated with oxidative phosphorylation, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, and the bacterial secretion system, showed a marked decline in the UC group compared with their spouses, but did not vary between healthy couples. Conclusion: Our study revealed that cohabitation with UC patients decreased differences in the gut microbiome between healthy individuals and patients. Not only was the composition and diversity of the microbiota diminished, but active pathways also showed some decline. Furthermore, Firmicutes, Faecalibacterium, and the four related pathways may be associated with the pathological state of the host rather than with human behavior.

Salmonella exploits membrane reservoirs for invasion of host cells

Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.

Progress toward a vaccine for extraintestinal pathogenic E. coli (ExPEC) II: efficacy of a toxin-autotransporter dual antigen approach.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of worldwide morbidity and mortality, the top cause of antimicrobial-resistant (AMR) infections, and the most frequent cause of life-threatening sepsis and urinary tract infections (UTI) in adults. The development of an effective and universal vaccine is complicated by this pathogen's pan-genome, its ability to mix and match virulence factors and AMR genes via horizontal gene transfer, an inability to decipher commensal from pathogens, and its intimate association and co-evolution with mammals. Using a pan virulome analysis of >20,000 sequenced E. coli strains, we identified the secreted cytolysin α-hemolysin (HlyA) as a high priority target for vaccine exploration studies. We demonstrate that a catalytically inactive pure form of HlyA, expressed in an autologous host using its own secretion system, is highly immunogenic in a murine host, protects against several forms of ExPEC infection (including lethal bacteremia), and significantly lowers bacterial burdens in multiple organ systems. Interestingly, the combination of a previously reported autotransporter (SinH) with HlyA was notably effective, inducing near complete protection against lethal challenge, including commonly used infection strains ST73 (CFT073) and ST95 (UTI89), as well as a mixture of 10 of the most highly virulent sequence types and strains from our clinical collection. Both HlyA and HlyA-SinH combinations also afforded some protection against UTI89 colonization in a murine UTI model. These findings suggest recombinant, inactive hemolysin and/or its combination with SinH warrant investigation in the development of an E. coli vaccine against invasive disease.

The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System

Bacterial surface display platforms have been developed for applications such as vaccine delivery and peptide library screening. The type V secretion system is an attractive anchoring motif for the surface expression of foreign proteins in gram-negative bacteria. SadA belongs to subtype C of the type V secretion system derived from Salmonella spp. and promotes biofilm formation and host cell adherence. The inner membrane lipoprotein SadB is important for SadA translocation. In this study, SadA was used as an anchoring motif to expose heterologous proteins in Salmonella typhimurium using SadB. The ability of SadA to display heterologous proteins on the S. typhimurium surface in the presence of SadB was approximately three-fold higher than that in its absence of SadB. Compared to full-length SadA, truncated SadAs (SadA877 and SadA269) showed similar display capacities when exposing the B-cell epitopes of urease B from Helicobacter pylori (UreB158–172aa and UreB349–363aa). We grafted different protein domains, including mScarlet (red fluorescent protein), the urease B fragment (UreBm) from H. pylori SS1, and/or protective antigen domain 4 from Bacillus anthracis A16R (PAD4), onto SadA877 or SadA1292. Whole-cell dot blotting, immunofluorescence, and flow cytometric analyses confirmed the localization of Flag×3-mScarlet (~30 kDa) and Flag×3-UreBm-mScarlet (~58 kDa) to the S. typhimurium surface using truncated SadA877 or SadA1292 as an anchoring motif. However, Flag×3-UreBm-PAD4-mScarlet (~75 kDa) was displayed on S. typhimurium using SadA1292. The oral administrated pSadBA1292-FUM/StmΔygeAΔmurI and pSadBA877-FUM/StmΔygeAΔmurI could elicit a significant mucosal and humoral immunity response. SadA could thus be used as an anchoring motif for the surface expression of large heterologous proteins as a potential strategy for attenuated bacterial vaccine development.

Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood–brain barrier damage in ducks during Riemerella anatipestifer infection

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

A comparison of DNA extraction methods used in the direct molecular detection of Burkholderia pseudomallei from blood.

Melioidosis is caused by Burkholderia pseudomallei. Direct molecular detection from unamplified blood remains insensitive. Three different extraction methods-QIAamp UCP Pathogen Mini Kit, High Pure PCR template and MagNA Pure Pathogen Universal-were trialled using spiked human ethylenediaminetetraacetic acid blood. A type III secretion system 1 (TTSS-1) polymerase chain reaction was used for detection. The QIAamp UCP Pathogen Mini Kit performed best, with a limit of detection of 1.5×102cfu/ml. It is planned to use the QIAamp UCP Pathogen Mini Kit to do a larger study on blood collected from patients with melioidosis.

Purification of Photorhabdus Virulence Cassette (PVC) Protein Complexes from Escherichia coli for Artificial Translocation of Heterologous Cargo Proteins.

Contractile injection systems (CISs), one of the most important bacterial secretion systems that transport substrates across the membrane, are a collection of diverse but evolutionarily related macromolecular devices. Numerous effector proteins can be loaded and injected by this secretion complex to their specific destinations. One group of CISs called extracellular CIS (eCIS) has been proposed as secretory molecules that can be released from the bacterial cytoplasm and attack neighboring target cells from the extracellular environment. This makes them a potential delivery vector for the transportation of various cargos without the inclusion of bacterial cells, which might elicit certain immunological responses from hosts. We have demonstrated that the Photorhabdus virulence cassette (PVC), which is a typical eCIS, could be applied as an ideal vector for the translocation of proteinaceous cargos with different physical or chemical properties. Here, we describe the in-depth purification protocol of this mega complex from Escherichia coli. The protocol provided is a simpler, faster, and more productive way of generating the eCIS complexes than available methodologies reported previously, which can facilitate the subsequent applications of these nanodevices and other eCIS in different backgrounds.

In vivo single-cell high-dimensional mass cytometry analysis to track the interactions between Klebsiella pneumoniae and myeloid cells.

In vivo single-cell approaches have transformed our understanding of the immune populations in tissues. Mass cytometry (CyTOF), that combines the resolution of mass spectrometry with the ability to conduct multiplexed measurements of cell molecules at the single cell resolution, has enabled to resolve the diversity of immune cell subsets, and their heterogeneous functionality. Here we assess the feasibility of taking CyTOF one step further to immuno profile cells while tracking their interactions with bacteria, a method we term Bac-CyTOF. We focus on the pathogen Klebsiella pneumoniae interrogating the pneumonia mouse model. Using Bac-CyTOF, we unveil the atlas of immune cells of mice infected with a K. pneumoniae hypervirulent strain. The atlas is characterized by a decrease in the populations of alveolar and monocyte-derived macrophages. Conversely, neutrophils, and inflammatory monocytes are characterized by an increase in the subpopulations expressing markers of less active cells such as the immune checkpoint PD-L1. These are the cells infected. We show that the type VI secretion system (T6SS) contributes to shape the lung immune landscape. The T6SS governs the interaction with monocytes/macrophages by shifting Klebsiella from alveolar macrophages to interstitial macrophages and limiting the infection of inflammatory monocytes. The lack of T6SS results in an increase of cells expressing markers of active cells, and a decrease in the subpopulations expressing PD-L1. By probing Klebsiella, and Acinetobacter baumannii strains with limited ability to survive in vivo, we uncover that a heightened recruitment of neutrophils, and relative high levels of alveolar macrophages and eosinophils and the recruitment of a characteristic subpopulation of neutrophils are features of mice clearing infections. We leverage Bac-CyTOF-generated knowledge platform to investigate the role of the DNA sensor STING in Klebsiella infections. sting-/- infected mice present features consistent with clearing the infection including the reduced levels of PD-L1. STING absence facilitates Klebsiella clearance.

Investigating the impact of insertion sequences and transposons in the genomes of the most significant phytopathogenic bacteria.

Genetic variability in phytopathogens is one of the main problems encountered for effective plant disease control. This fact may be related to the presence of transposable elements (TEs), but little is known about their role in host genomes. Here, we performed the most comprehensive analysis of insertion sequences (ISs) and transposons (Tns) in the genomes of the most important bacterial plant pathogens. A total of 35 692 ISs and 71 transposons were identified in 270 complete genomes. The level of pathogen-host specialization was found to be a significant determinant of the element distribution among the species. Some Tns were identified as carrying virulence factors, such as genes encoding effector proteins of the type III secretion system and resistance genes for the antimicrobial streptomycin. Evidence for IS-mediated ectopic recombination was identified in Xanthomonas genomes. Moreover, we found that IS elements tend to be inserted in regions near virulence and fitness genes, such ISs disrupting avirulence genes in X. oryzae genomes. In addition, transcriptome analysis under different stress conditions revealed differences in the expression of genes encoding transposases in the Ralstonia solanacearum, X. oryzae, and P. syringae species. Lastly, we also investigated the role of Tns in regulation via small noncoding regulatory RNAs and found these elements may target plant-cell transcriptional activators. Taken together, the results indicate that TEs may have a fundamental role in variability and virulence in plant pathogenic bacteria.

Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella’s intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell–cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

A sophisticated virulence repertoire and colistin resistance of Citrobacter freundii ST150 from a patient with sepsis admitted to ICU in a tertiary care hospital in Uganda, East Africa: Insight from genomic and molecular docking analyses

Sepsis and multidrug resistance comprise a complex of factors attributable to mortality among intensive care unit (ICU) patients globally. Pathogens implicated in sepsis are diverse, and their virulence and drug resistance remain elusive. From a tertiary care hospital ICU in Uganda, we isolated a Citrobacter freundii strain RSM030 from a patient with sepsis and phenotypically tested it against a panel of 16 antibiotics including imipenem levofloxacin, cotrimoxazole and colistin, among others. We sequenced the organism's genome and integrated multilocus sequencing (MLST), PathogenFinder with Virulence Factor analyzer (VFanalyzer) to establish its pathogenic relevance. Thereafter, we combined antiSMASH and PRISM genome mining with molecular docking to predict biosynthetic gene clusters (BGCs), pathways, toxin structures and their potential targets in-silico. Finally, we coupled ResFinder with comprehensive antibiotic resistance database (CARD) to scrutinize the genomic antimicrobial resistance profile of the isolate. From PathogenFinder and MLST, this organism was confirmed to be a human pathogen (p = 0.843), sequence type (ST)150, whose virulence is determined by chromosomal type III secretion system (T3SS) (the injectosome) and plasmid-encoded type IV secretion system (T4SS), the enterobactin biosynthetic gene cluster and biofilm formation through the pgaABCD operon. Pathway and molecular docking analyses revealed that the shikimate pathway can generate a toxin targeting multiple host proteins including spectrin, detector of cytokinesis protein 2 (Dock2) and plasmalemma vesicle-associated protein (PLVAP), potentially distorting the host cell integrity. From phenotypic antibiotic testing, we found indeterminate results for amoxicillin/clavulanate and levofloxacin, with resistance to cotrimoxazole and colistin. Detailed genome analysis revealed chromosomal beta lactam resistance genes, i.e. blaCMY-79, blaCMY-116 and blaTEM-1B, along with multiple mutations of the lipopolysaccharide modifying operon genes PmrA/PmrB, pmrD, mgrA/mgrB and PhoP/PhoQ, conferring colistin resistance. From these findings, we infer that Citrobacter freundii strain RSM030 is implicated in sepsis and resistance to standard antibiotics, including colistin, the last resort.

Transcriptome analysis reveals the inhibitory mechanism of 3,4-Dimethoxyphenol from Streptomyces albidoflavus strain ML27 against Xanthomonas oryzae pv. oryzae

Bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), poses a significant threat to rice cultivation across diverse regions. Growing concerns about pesticide resistance and environmental impact underscore the urgent necessity for eco-friendly biopesticides. Here, the complete genome sequence of Streptomyces albidoflavus strain ML27 revealed substantial antimicrobial activity and secondary metabolite production potential through genome mining. 3,4-dimethoxyphenol (purity 97%) was successfully isolated from the fermentation broth of S. albidoflavus strain ML27, exhibiting broad and pronounced inhibitory effects on the growth of seven different fungi and five tested bacteria. The efficacy of 3,4-dimethoxyphenol in controlling rice bacterial leaf blight was evaluated through pot tests, demonstrating substantial therapeutic (69.39%) and protective (84.53%) effects. Application of 3,4-dimethoxyphenol to Xoo resulted in cells displayed notable surface depressions, wrinkles, distortions, or even ruptures compared to their typical morphology. Transcriptome analysis revealed significant inhibition of membrane structures, protein synthesis and secretion, bacterial secretion system, two-component system, flagellar assembly, as well as various metabolic and biosynthetic pathways by 3,4-dimethoxyphenol. Notably, the down-regulation of the type III secretion system (T3SS) expression was a pivotal finding. Furthermore, validation via quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed significant downregulation of 10 genes related to T3SS upon 3,4-dimethoxyphenol treatment. Based on these results, it is promising to develop 3,4-dimethoxyphenol as a novel biopesticide targeting the T3SS of Xoo for controlling bacterial leaf blight in rice.

A hypervirulent Acinetobacter baumannii strain has robust anti-phagocytosis ability

BackgroundAcinetobacter baumannii (A. baumannii) is associated with both hospital-acquired infections (HAP) and community-acquired pneumonia (CAP). In this study, we present a novel CAP-associated A. baumannii (CAP-AB) strain causing severe pneumonia in an afore healthy male patient without underlying conditions. Subsequently, we investigated the pathogenicity and immunogenicity of this CAP-AB strain using a mice pneumonia model.ResultsA 58-year-old male patient with no underlying conditions experienced worsening symptoms of a productive cough, sputum, and fever that developed acutely, in just 24 h. The diagnosis was severe community-acquired pneumonia (CAP) and type-1 respiratory failure. An A. baumannii strain was isolated from his sputum and blood cultures. To gain a deeper understanding of the rapid progression of its pathology, we utilized the CAP-associated A. baumannii strain YC128, a previously obtained hospital-acquired pneumonia A. baumannii (HAP-AB) strain YC156, and a highly virulent A. baumannii control strain LAC-4 to construct a mouse pneumonia model, and subsequently compared the mortality rate of the three groups. Following inoculation with 107 CFU of A. baumannii, the mortality rate for the YC128, LAC-4, and YC156 groups was 60% (6/10), 30% (3/10), and 0%, respectively. The bacterial burden within the pulmonary, liver, and spleen tissues of mice in the YC128 group was significantly higher than that of the YC156 group, and slightly higher than that of the LAC-4 group. Pathological analysis of lung tissue using HE-staining revealed that the inflammatory pathological changes in mice from the YC128 group were significantly more severe than those in the YC156 group. Additionally, CT scan images displayed more pronounced inflammation in the lungs of mice from the YC128 group compared to the YC156 group. Local levels of cytokines/chemokines such as IL-1β, IL-6, TNF-α, and CXCL1 were assessed via RT-qPCR in lung tissues. In comparison with the YC156 strain, the highly virulent YC128 strain induced the expression of proinflammatory cytokines more rapidly and severely. Furthermore, we examined the in vitro anti-phagocytosis ability of YC128 and YC156 strains against mice peritoneal macrophages, revealing that the highly virulent YC128 isolate displayed greater resistance to macrophage uptake in contrast to YC156. Results from Whole Genome Sequencing (WGS) indicated that YC128 harbored a complete type VI secretion system (T6SS) gene cluster, while YC156 lacked the majority of genes within the T6SS gene cluster. The other virulence-related genes exhibited minimal differences between YC128 and YC156. Drawing from previous studies, we postulated that the T6SS is linked to the hypervirulence and robust anti-phagocytic ability of YC128.ConclusionsThis article reports on the isolation of a novel hypervirulent CAP-AB strain, YC128, from a severe CAP patient. The results demonstrate that this CAP-AB strain, YC128, is capable of inducing fatal pneumonia and extrapulmonary dissemination in a mouse pneumonia model. Moreover, this highly virulent CAP-AB strain exhibits significantly stronger anti-phagocytic abilities compared to the HAP-AB YC156 strain. Genome sequencing comparisons reveal that the heightened hypervirulence and enhanced anti-phagocytosis abilities observed in YC128 may be attributed to the presence of the T6SS.

Pectobacterium araliae sp. nov., a pathogen causing bacterial soft rot of Japanese angelica tree in Japan.

Phytopathogenic bacteria (MAFF 302110T and MAFF 302107) were isolated from lesions on Japanese angelica trees affected by bacterial soft rot in Yamanashi Prefecture, Japan. The strains were Gram-reaction-negative, facultatively anaerobic, motile with peritrichous flagella, rod-shaped, and non-spore-forming. The genomic DNA G+C content was 51.1 mol % and the predominant cellular fatty acids included summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 2 (comprising any combination of C12 : 0 aldehyde, an unknown fatty acid with an equivalent chain length of 10.928, C16 : 1 iso I, and C14 : 0 3OH), and C12 : 0. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences, along with phylogenomic analysis utilizing whole-genome sequences, consistently placed these strains within the genus Pectobacterium. However, their phylogenetic positions did not align with any known species within the genus. Comparative studies involving average nucleotide identity and digital DNA-DNA hybridization with the closely related species indicated values below the thresholds employed for the prokaryotic species delineation (95-96 % and 70 %, respectively), with the highest values observed for Pectobacterium polonicum DPMP315T (92.10 and 47.1 %, respectively). Phenotypic characteristics, cellular fatty acid composition, and a repertoire of secretion systems could differentiate the strains from their closest relatives. The phenotypic, chemotaxonomic, and genotypic data obtained in this study show that MAFF 302110T/MAFF 302107 represent a novel species of the genus Pectobacterium, for which we propose the name Pectobacterium araliae sp. nov., designating MAFF 302110T (=ICMP 25161T) as the type strain.

Class II diacylglycerol kinases participate in the basal immune responses of Nicotiana benthamiana to Ralstonia solanacearum

Phospholipid signaling plays an important role in Nicotiana benthamiana immune responses to phytopathogenic bacteria. In this study, we isolated three orthologs encoding class II diacylglycerol kinase (NbDGK4-1, NbDGK4-2 and NbDGK7) in the N. benthamiana genome. In contrast to the undetectable expression of NbDGK4-2, the NbDGK4-1 and NbDGK7 expression levels increased in response to Ralstonia solanacearum and the flg22 peptide. The induction of the hypersensitive response was not affected, but bacterial growth increased and the onset of bacterial wilt symptoms was accelerated in the NbDGK4-1-silenced and NbDGK7-silenced plants challenged with R. solanacearum. The expression levels of NbPR-1 and NbPR-4 (marker genes for salicylic acid and jasmonic acid signaling, respectively) decreased in the NbDGK4-1-silenced and NbDGK7-silenced plants inoculated with R. solanacearum. The expression of pathogen-associated molecular pattern-triggered immunity (PTI) marker genes were also inhibited in the NbDGK4-1-silenced and NbDGK7-silenced plants infiltrated with the type III secretion system (T3SS)-deficient R. solanacearum mutant or flg22. Accordingly, the T3SS-deficient R. solanacearum bacterial population increased in the NbDGK4-1-silenced and NbDGK7-silenced plants. Moreover, flg22-induced resistance and callose deposition were also compromised in the NbDGK4-1-silenced and NbDGK7-silenced plants. Considered together, the study results indicate class II diacylglycerol kinases may be important for N. benthamiana basal disease resistance (e.g., PTI responses).