mBio最新文献

{"title":"The pneumococcal bacteriocin streptococcin B is produced as part of the early competence cascade and promotes intraspecies competition.","authors":"J D Richardson, Emily Guo, Ryan M Wyllie, Paul Jensen, Suzanne Dawid","doi":"10.1128/mbio.02993-24","DOIUrl":"https://doi.org/10.1128/mbio.02993-24","url":null,"abstract":"<p><p><i>Streptococcus pneumoniae</i> is an important human pathogen that normally resides in the human nasopharynx. Competence-mediated bacteriocin expression by <i>S. pneumoniae</i> plays a major role in both the establishment and persistence of colonization on this polymicrobial surface. Over 20 distinct bacteriocin loci have been identified in pneumococcal genomes, but only a small number have been characterized phenotypically. In this work, we demonstrate that three-fourths of <i>S. pneumoniae</i> strains contain a highly conserved <i>scb</i> locus that encodes an active lactococcin 972-like bacteriocin called streptococcin B. In these backgrounds, the <i>scbABC</i> locus is part of the early competence cascade due to a ComE binding site in the promoter region. Streptococcin B producing strains target both members of the population that have failed to activate competence and the 25% of the population that carry a naturally occurring deletion of the ComE binding site and the functional bacteriocin gene. The ComR-type regulator found directly upstream of the <i>scb</i> locus in <i>S. pneumoniae</i> strains can activate <i>scb</i> expression independent of the presence of the ComE binding site but only when stimulated by a peptide that is encoded in the <i>scb</i> locus of <i>Streptococcus pseudopneumoniae</i>, a closely related bacterium that also inhabits the human nasopharynx. Given the co-regulation with competence and the phenotypic confirmation of activity, streptococcin B represents a previously unrecognized fratricide effector that gives producing strains an additional advantage over the naturally occurring deleted strains during colonization.</p><p><strong>Importance: </strong><i>Streptococcus pneumoniae</i> is a common cause of pneumonia, meningitis, sinusitis, and otitis media. In order to successfully colonize humans, a prerequisite to the development of invasive disease, <i>S. pneumoniae</i> must compete with other bacterial inhabitants of the nasal surface for space and nutrients. Bacteriocins are small antimicrobial peptides produced by bacteria that typically target neighboring bacteria by disruption of the cell surface. <i>S. pnuemoniae</i> encodes a large number of potential bacteriocin, but, for most, their role in competitive interactions has not been defined. This work demonstrates that isolates that produce the bacteriocin streptococcin B have an advantage over non-producers. These observations contribute to our understanding of the competitive interactions that precede the development of <i>S. pneumoniae</i> disease.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0299324"},"PeriodicalIF":5.1,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142837464","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of a novel <i>covS</i> SNP identified in Australian group A <i>Streptococcus</i> isolates derived from the M1_UK lineage.","authors":"Johanna Richter, Amanda J Cork, Yvette Ong, Nadia Keller, Andrew J Hayes, Mark A Schembri, Amy V Jennison, Mark R Davies, Kate Schroder, Mark J Walker, Stephan Brouwer","doi":"10.1128/mbio.03366-24","DOIUrl":"https://doi.org/10.1128/mbio.03366-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Group A &lt;i&gt;Streptococcus&lt;/i&gt; (GAS) is a human-adapted pathogen responsible for a variety of diseases. The GAS M1&lt;sub&gt;UK&lt;/sub&gt; lineage has contributed significantly to the recently reported increases in scarlet fever and invasive infections. However, the basis for its evolutionary success is not yet fully understood. During the transition to systemic disease, the M1 serotype is known to give rise to spontaneous mutations in the control of virulence two-component regulatory system (CovRS) that confer a fitness advantage during invasive infections. Mutations that inactivate CovS function result in the de-repression of key GAS virulence factors such as streptolysin O (SLO), a pore-forming toxin and major trigger of inflammasome/interleukin-1β-dependent inflammation. Conversely, expression of the streptococcal cysteine protease SpeB, which is required during initial stages of colonization and onset of invasive disease, is typically lost in such mutants. In this study, we identified and characterized a novel &lt;i&gt;covS&lt;/i&gt; single nucleotide polymorphism detected in three separate invasive M1&lt;sub&gt;UK&lt;/sub&gt; isolates. The resulting CovS&lt;sup&gt;Ala318Val&lt;/sup&gt; mutation caused a significant upregulation of SLO resulting in increased inflammasome activation in human THP-1 macrophages, indicating an enhanced inflammatory potential. Surprisingly, SpeB production was unaffected. Site-directed mutagenesis was performed to assess the impact of this mutation on virulence and global gene expression. We found that the CovS&lt;sup&gt;Ala318Val&lt;/sup&gt; mutation led to subtle, virulence-specific changes of the CovRS regulon compared to previously characterized &lt;i&gt;covS&lt;/i&gt; mutations, highlighting an unappreciated level of complexity in CovRS-dependent gene regulation. Continued longitudinal surveillance is warranted to determine whether this novel &lt;i&gt;covS&lt;/i&gt; mutation will expand in the M1&lt;sub&gt;UK&lt;/sub&gt; lineage.IMPORTANCEThe M1&lt;sub&gt;UK&lt;/sub&gt; lineage of GAS has contributed to a recent global upsurge in scarlet fever and invasive infections. Understanding how GAS can become more virulent is critical for infection control and identifying new treatment approaches. The two-component CovRS system, comprising the sensor kinase CovS and transcription factor CovR, is a central regulator of GAS virulence genes. In the M1 serotype, &lt;i&gt;covRS&lt;/i&gt; mutations are associated with an invasive phenotype. Such mutations have not been fully characterized in the M1&lt;sub&gt;UK&lt;/sub&gt; lineage. This study identified a novel &lt;i&gt;covS&lt;/i&gt; mutation in invasive Australian M1&lt;sub&gt;UK&lt;/sub&gt; isolates that resulted in a more nuanced virulence gene regulation compared to previously characterized &lt;i&gt;covS&lt;/i&gt; mutations. A representative isolate displayed upregulated SLO production and triggered amplified interleukin-1β secretion in infected human macrophages, indicating an enhanced inflammatory potential. These findings underscore the need for comprehensive analyses of &lt;i&gt;covRS&lt;/i&gt; mutants to fully elucidate their contribu","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0336624"},"PeriodicalIF":5.1,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142837241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stage-specific function of sphingolipid synthases in African trypanosomes.","authors":"Norton Heise, Carolina M Koeller, Mohamed Sharif, James D Bangs","doi":"10.1128/mbio.03501-24","DOIUrl":"https://doi.org/10.1128/mbio.03501-24","url":null,"abstract":"<p><p>The protozoan parasite <i>Trypanosoma brucei</i> is the only known eukaryote capable of synthesizing the three main phosphosphingolipids: sphingomyelin (SM), inositol phosphorylceramide (IPC), and ethanolamine phosphorylceramide (EPC). It has four paralogous genes encoding sphingolipid synthases (<i>TbSLS1-4</i>). TbSLS1 is a dedicated IPC synthase, TbSLS2 is a dedicated EPC synthase, and TbSLS3 and TbSLS4 are bifunctional SM/EPC synthases. IPC synthesis occurs exclusively in the procyclic insect stage (PCF), EPC is limited to the mammalian bloodstream form (BSF), and SM is synthesized throughout the life cycle. TbSLSs are indispensable for the viability of BSF and are, thus, potential drug targets. The relative stage-specific expression of each <i>TbSLS</i> paralog was compared, and the results match phosphosphingolipid content. Induction of pan-specific RNAi silencing was lethal in both BSF and PCF. To investigate individual TbSLS functions, separate HA-tagged genes, recoded to be RNAi-resistant (RNAi^R), were engineered to replace a single allele of the entire <i>TbSLS</i> locus within parental BSF and PCF RNAi cell lines. RNAi^R <i>TbSLS3</i> and <i>TbSLS4</i> both rescued BSF growth under silencing. Expression of RNAi^R <i>TbSLS1</i>, normally repressed in BSF, did not rescue BSF viability but was not detrimental to normal <i>in vitro</i> growth. RNAi^R <i>TbSLS1</i>, <i>TbSLS3</i>, and <i>TbSLS4</i> were each sufficient to rescue PCF growth, indicating IPC is not essential for PCF viability <i>in vitro</i>. All TbSLSs localize to distal Golgi compartments in both BSF and PCF cells. These findings raise interesting questions about the roles of individual phosphosphingolipids in <i>in vivo</i> infection of the mammalian and tsetse hosts.</p><p><strong>Importance: </strong>African trypanosomes are eukaryotic pathogens that cause human and veterinary African trypanosomaisis. Uniquely, they synthesize all three major phosphosphingolipid species using four distinct sphingolipid synthases (SLS). This work details the function of each SLS in both bloodstream and insect form parasites. Novel and unexpected sphingolipid dependences are found in each stage. These results are consistent with this metabolic pathway being a valid target for chemotherapeutic intervention.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0350124"},"PeriodicalIF":5.1,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142829245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The novel H10N3 avian influenza virus acquired airborne transmission among chickens: an increasing threat to public health.","authors":"Xiaoquan Wang, Huiyan Yu, Yahao Ma, Pinghu Zhang, Xiyue Wang, Jianyu Liang, Xiuling Zhang, Ruyi Gao, Xiaolong Lu, Wenhao Yang, Yu Chen, Min Gu, Jiao Hu, Xiaowen Liu, Shunlin Hu, Daxin Peng, Xian Qi, Changjun Bao, Kaituo Liu, Xiufan Liu","doi":"10.1128/mbio.02363-24","DOIUrl":"https://doi.org/10.1128/mbio.02363-24","url":null,"abstract":"<p><p>Following two human infections with the H10N3 avian influenza virus (AIV) in 2021 and 2022, a third case was discovered in Yunnan, China, in 2024, raising concerns about the potential for future pandemics. Recent studies have indicated that novel H10N3 viruses are highly pathogenic in mice and can be transmitted between guinea pigs via respiratory droplets without prior adaptation. However, the biological characteristics of novel H10N3 in poultry have not been fully elucidated. Our findings revealed that H10 subtype AIVs are predominantly prevalent in waterfowl. Notably, H10N8 and H10N3 viruses that have infected humans were primarily isolated from chickens. For the first time, double basic hemagglutinin cleavage sites (motif PEIKQGR↓GL) were identified in novel H10N3 AIVs, which exhibit enhanced replication in chickens, and can be transmitted between chickens through direct contact and respiratory droplets. Animal experimental studies demonstrated that ducks are also susceptible to H10N3 viruses and that the virus is transmissible through direct contact, suggesting a greater risk of transmission and recombination. Serological studies conducted among poultry workers suggest that while the human population was largely naïve to H10N3 infection, sporadic and undetected human infections did occur, indicating a potential increasing trend. These data further emphasize the growing threat to public health posed by zoonotic H10N3 subtype AIVs.IMPORTANCEExposure to poultry in live poultry markets (LPMs) is strongly associated with human infection with avian influenza viruses (AIVs), with chickens being the most common species found in these markets in China. The prevalence of AIVs in chickens, therefore, increases the risk of human infection. Notably, the main host of the novel H10N3 virus has shifted from waterfowl to chickens, and the virus can be transmitted between chickens via respiratory droplets, posing a potential risk of a pandemic within poultry populations. The novel H10N3 virus also remains sensitive to ducks and can be transmitted through direct contact, which means a greater risk of transmission and recombination. Significantly, the human population remains largely naïve to H10N3 infection, but sporadic seropositivity among poultry workers indicates previous exposure to H10 subtype AIVs. Therefore, a comprehensive surveillance of the novel H10N3 viruses in poultry is imperative. Effective control of the virus within poultry populations could significantly reduce the risk of emerging human infections.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0236324"},"PeriodicalIF":5.1,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142829247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inhibition and evasion of neutrophil microbicidal responses by <i>Legionella longbeachae</i>.","authors":"Hannah E Hanford, Christopher T D Price, Silvia Uriarte, Yousef Abu Kwaik","doi":"10.1128/mbio.03274-24","DOIUrl":"https://doi.org/10.1128/mbio.03274-24","url":null,"abstract":"<p><p><i>Legionella</i> species evade degradation and proliferate within alveolar macrophages as an essential step for the manifestation of disease. However, most intracellular bacterial pathogens are restricted in neutrophils, which are the first line of innate immune defense against invading pathogens. Bacterial degradation within neutrophils is mediated by the fusion of microbicidal granules to pathogen-containing phagosomes and the generation of reactive oxygen species (ROS) by the phagocyte NADPH oxidase complex. Here, we show that human neutrophils fail to trigger microbicidal processes and, consequently, fail to restrict <i>L. longbeachae</i>. In addition, neutrophils infected with <i>L. longbeachae</i> fail to undergo a robust pro-inflammatory response, such as degranulation and IL-8 production. Here, we identify three strategies employed by <i>L. longbeachae</i> for evading restriction by neutrophils and inhibiting the neutrophil microbicidal response to other bacteria co-inhabiting in the same cell. First, <i>L. longbeachae</i> excludes the cytosolic and membrane-bound subunits of the phagocyte NADPH oxidase complex from its phagosomal membrane independent of the type 4 secretion system (T4SS). Consequently, infected neutrophils fail to generate robust ROS in response to <i>L. longbeachae</i>. Second, <i>L. longbeachae</i> impedes the fusion of azurophilic granules to its phagosome and the phagosomes of bacteria co-inhabiting the same cell through T4SS-independent mechanisms. Third, <i>L. longbeachae</i> protects phagosomes of co-inhabiting bacteria from degradation by ROS through a <i>trans</i>-acting T4SS-dependent mechanism. Collectively, we conclude that <i>L. longbeachae</i> evades restriction by human neutrophils <i>via</i> T4SS-independent mechanisms and utilizes <i>trans</i>-acting T4SS-dependent mechanisms for inhibition of neutrophil ROS generation throughout the cell cytosol.</p><p><strong>Importance: </strong><i>Legionella longbeachae</i> is commonly found in soil environments where it interacts with a wide variety of protist hosts and microbial competitors. Upon transmission to humans<i>, L. longbeachae</i> invades and replicates within alveolar macrophages, leading to the manifestation of pneumonia. In addition to alveolar macrophages, neutrophils are abundant immune cells acting as the first line of defense against invading pathogens. While most intracellular bacterial species are killed and degraded by neutrophils, we show that <i>L. longbeachae</i> evades degradation. The pathogen impairs the major neutrophils' microbicidal processes, including the fusion of microbicidal granules to the pathogen-containing vacuole. By inhibiting of assembly of the phagocyte NADPH oxidase complex, the pathogen blocks neutrophils from generating microbicide reactive oxygen species. Overall, <i>L. longbeachae</i> employs unique virulence strategies to evade the major microbicidal processes of neutrophils.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0327424"},"PeriodicalIF":5.1,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142829215","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Overcoming barriers for investigating nickel-pincer nucleotide cofactor-related enzymes.","authors":"Jorge L Nevarez, Aiko Turmo, Santhosh Gatreddi, Swati Gupta, Jian Hu, Robert P Hausinger","doi":"10.1128/mbio.03404-24","DOIUrl":"https://doi.org/10.1128/mbio.03404-24","url":null,"abstract":"<p><p>The nickel-pincer nucleotide (NPN) cofactor is a modified pyridinium mononucleotide that tri-coordinates nickel and is crucial for the activity of certain racemases and epimerases. LarB, LarC, and LarE are responsible for NPN synthesis, with the cofactor subsequently installed into LarA homologs. Hurdles for investigating the functional properties of such proteins arise from the difficulty of obtaining the active, NPN cofactor-loaded enzymes and in assaying their diverse reactivities. Here, we show that when the <i>Lactiplantibacillus plantarum lar</i> genes are cloned into the Duet expression system and cultured in <i>Escherichia coli</i>, they confer lactate racemase activity to the cells. By replacing <i>L. plantarum larA</i> with related genes from other microorganisms, this system allows for the generation of active LarA homologs. Furthermore, the Duet system enables the functional testing of LarB, LarC, and LarE homologs from other microorganisms. In addition to applying the Duet expression system for synthesis of active, NPN cofactor-containing enzymes in <i>E. coli</i>, we demonstrate that circular dichroism spectroscopy provides a broadly applicable means of assaying these enzymes. By selecting a wavelength of high molar ellipticity and low absorbance for a given 2-hydroxy acid substrate enantiomer, the conversion of one enantiomer/epimer into the other can be monitored for LarA homologs without the need for any coupling enzymes or reagents. The methods discussed here further our abilities to investigate the unique activities of Lar proteins.</p><p><strong>Importance: </strong>Enzymes containing the nickel-pincer nucleotide (NPN) cofactor are prevalent in a wide range of microorganisms and catalyze various critical biochemical reactions, yet they remain underexplored due, in part, to limitations in current research methodologies. The two significant advancements described here, the heterologous production of active NPN-cofactor containing enzymes in <i>Escherichia coli</i> and the use of a circular dichroism-based assay to monitor enzyme activities, expand our capacity to analyze these enzymes. Such additional detailed characterization will deepen our understanding of the diverse chemistry catalyzed by the NPN cofactor and potentially uncover novel roles for this organometallic species in microbial metabolism.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0340424"},"PeriodicalIF":5.1,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142829219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The <i>Campylobacter jejuni</i> BumS sensor phosphatase detects the branched short-chain fatty acids isobutyrate and isovalerate as direct cues for signal transduction.","authors":"Nestor Ruiz, Jiawei Xing, Igor B Zhulin, Chad A Brautigam, David R Hendrixson","doi":"10.1128/mbio.03278-24","DOIUrl":"https://doi.org/10.1128/mbio.03278-24","url":null,"abstract":"<p><p>Two-component signal transduction systems (TCSs) are nearly ubiquitous across bacterial species and enable bacteria to sense and respond to specific cues for environmental adaptation. The <i>Campylobacter jejuni</i> BumSR TCS is unusual in that the BumS sensor exclusively functions as a phosphatase rather than a kinase to control phosphorylated levels of its cognate BumR response regulator (P-BumR). We previously found that BumSR directs a response to the short-chain fatty acid butyrate generated by resident microbiota so that <i>C. jejuni</i> identifies ideal lower intestinal niches in avian and human hosts for colonization. However, butyrate is an indirect cue for BumS and did not inhibit <i>in vitro</i> BumS phosphatase activity for P-BumR. In this work, we expanded the repertoire of lower intestinal metabolites that are cues sensed by BumS that modulate the expression of genes required for colonization to include the branched short-chain fatty acids isobutyrate and isovalerate. Unlike butyrate, isobutyrate and isovalerate inhibited <i>in vitro</i> BumS phosphatase activity for P-BumR, indicating that these metabolites are direct cues for BumS. Isobutyrate and isovalerate reduced the thermostability of BumS and caused a reorganization of protein structure to suggest how sensing these cues inhibits phosphatase activity. We also identified residues in the BumS sensory domain required to detect isobutyrate, isovalerate, and butyrate and for optimal colonization of hosts to reveal how gut bacteria can recognize these intestinal metabolites. Our work reveals how this unusual bacterial sensor phosphatase senses a repertoire of intestinal metabolites and how cues alter BumSR signal transduction to influence <i>C. jejuni</i> colonization of hosts.IMPORTANCETCSs are prevalent in many bacteria, but the cues sensed by each are not actually known for many of these systems. Microbiota-generated butyrate in human and avian hosts is detected by the <i>Campylobacter jejuni</i> BumS sensor phosphatase so that the bacterium identifies ideal lower intestinal niches for colonization. However, BumS only indirectly senses butyrate to inhibit dephosphorylation of its cognate BumR response regulator. Here, we expanded the repertoire of cues sensed by BumS to the branched-short chain fatty acids isobutyrate and isovalerate that are also abundant in the lower intestines. Both isobutyrate and isovalerate are potent, direct cues for BumS, whereas butyrate is an indirect cue. Leveraging isobutyrate and isovalerate as direct cues, we reveal BumS structure is altered upon cue detection to inhibit its phosphatase activity. We provide an understanding of the mechanics of an unusual mode of signal transduction executed by BumSR and other bacterial sensor phosphatase-driven TCSs.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0327824"},"PeriodicalIF":5.1,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142818558","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Gcn2 rescues reprogramming in the absence of Hog1/p38 signaling in <i>C. neoformans</i> during thermal stress.","authors":"David Goich, Amanda L M Bloom, Sean R Duffy, Maritza N Ventura, John C Panepinto","doi":"10.1128/mbio.01762-24","DOIUrl":"10.1128/mbio.01762-24","url":null,"abstract":"<p><p>The fungus <i>Cryptococcus neoformans</i> is an opportunistic pathogen of humans that reprograms its translatome to facilitate adaptation and virulence within the host. We studied the role of Hog1/p38 in reprogramming translation during thermal stress adaptation and found that this pathway acts on translation <i>via</i> crosstalk with the Gcn2 pathway, a well-studied regulator of general translation control. Using a combination of molecular assays and phenotypic analysis, we show that increased output from the Gcn2 pathway in a Hog1 deletion mutant is associated with rescue of thermal stress adaptation at both molecular and phenotypic scales. We characterize known outputs of the Hog1 pathway during thermal stress as either Gcn2-dependent or Gcn2-independent and demonstrate that Hog1 activation regulates the Gcn2 pathway even in the absence of thermal stress. Finally, we implicate this phenomenon in another Hog1-regulated process, morphogenesis, and recapitulate Hog1-Gcn2 crosstalk in the distantly related fungal pathogen, <i>Candida albicans</i>. Our results point to an important link between the stress response machinery and translation control and clarify the etiology of phenotypes associated with Hog1 deletion. More broadly, this study highlights complex interplay between core conserved signal transduction pathways and the utility of molecular assays to better understand how these pathways are connected.IMPORTANCE<i>Cryptococcus neoformans</i> is an opportunistic pathogen of humans that causes deadly cryptococcal meningitis, which is is responsible for an estimated 19% of AIDS-related mortality. When left untreated, cryptococcal meningitis is uniformly fatal, and in patients receiving the most effective antifungal regimens, mortality remains high. Thus, there is a critical need to identify additional targets that play a role in the adaptation to the human host and virulence. This study explores the role of the stress response kinases Hog1 and Gcn2 in thermoadaptation, which is a pre-requisite for virulence. Our results show that compensatory signaling occurs <i>via</i> the Gcn2 pathway when Hog1 is deleted, and that disruption of both pathways increases sensitivity to thermal stress. Importantly, our study highlights the insufficiency of using single-gene deletion mutants to study gene function, since many phenotypes associated with Hog1 deletion were driven by Gcn2 signaling in this background, rather than loss of direct Hog1 activity.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0176224"},"PeriodicalIF":5.1,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142818499","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of an sRNA-mediated conditional knockdown system for <i>Chlamydia trachomatis</i>.","authors":"Janina Ehses, Kevin Wang, Asha Densi, Cuper Ramirez, Ming Tan, Christine Sütterlin","doi":"10.1128/mbio.02545-24","DOIUrl":"10.1128/mbio.02545-24","url":null,"abstract":"<p><p>We describe a new <i>Chlamydia trachomatis</i> protein depletion method that uses an engineered small RNA (sRNA) to inhibit translation of a target gene. In proof-of-principle experiments, we induced functional knockdown of IncA, a fusion-mediating inclusion membrane protein, as shown with Western blots, loss of IncA staining at the inclusion membrane, and production of multiple chlamydial inclusions within an infected cell. These effects were titratable and reversible. To test for polar effects, we separately targeted the inclusion membrane proteins IncE and IncG, which are expressed from the <i>incDEFG</i> operon. Knockdown of IncE caused loss of IncE and its interacting host protein SNX6 at the inclusion membrane, without affecting IncG protein levels. Similarly, IncG knockdown significantly reduced IncG levels and prevented recruitment of its interacting host protein 14-3-3β, without altering IncE protein levels. These data provide the first genetic evidence that IncE and IncG are necessary for the recruitment of SNX6 and 14-3-3β, respectively, demonstrating the value of this knockdown approach. We also successfully depleted the major chlamydial surface protein, major outer membrane protein (MOMP), which is encoded by a likely essential gene that has not been previously disrupted or knocked down. MOMP knockdown caused severe defects in bacterial morphology and progeny production. Thus, our sRNA-based approach has broad potential as a conditional knockdown method for studying the function of <i>C. trachomatis</i> genes, including essential genes and genes in an operon.IMPORTANCEWe describe a new method to reduce protein levels of a selected gene in the pathogenic bacterium <i>Chlamydia trachomatis</i>. This approach utilizes an engineered small RNA (sRNA) to inhibit translation of the mRNA for a target gene and produced inducible and reversible protein knockdown. Our method successfully knocked down four proteins, including a likely essential gene and individual genes in an operon, without altering protein levels of a neighboring gene. This conditional knockdown method will be useful for studying the function of genes in <i>Chlamydia</i>. It also has the potential to be applied to other obligate intracellular bacteria, including <i>Rickettsia</i> and <i>Coxiella</i>.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0254524"},"PeriodicalIF":5.1,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142818470","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"OXA β-lactamases from <i>Acinetobacter</i> spp. are membrane bound and secreted into outer membrane vesicles.","authors":"Lucia Capodimonte, Fernando Teixeira Pinto Meireles, Guillermo Bahr, Robert A Bonomo, Matteo Dal Peraro, Carolina López, Alejandro J Vila","doi":"10.1128/mbio.03343-24","DOIUrl":"10.1128/mbio.03343-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;β-lactamases from Gram-negative bacteria are generally regarded as soluble, periplasmic enzymes. NDMs have been exceptionally characterized as lipoproteins anchored to the outer membrane. A bioinformatics study on all sequenced β-lactamases was performed that revealed a predominance of putative lipidated enzymes in the Class D OXAs. Namely, 60% of the OXA Class D enzymes contain a lipobox sequence in their signal peptide, that is expected to trigger lipidation and membrane anchoring. This contrasts with β-lactamases from other classes, which are predicted to be mostly soluble proteins. Almost all (&gt;99%) putative lipidated OXAs are present in &lt;i&gt;Acinetobacter&lt;/i&gt; spp. Importantly, we further demonstrate that OXA-23 and OXA-24/40 are lipidated, membrane-bound proteins in &lt;i&gt;Acinetobacter baumannii&lt;/i&gt;. In contrast, OXA-48 (commonly produced by Enterobacterales) lacks a lipobox and is a soluble protein. Outer membrane vesicles (OMVs) from &lt;i&gt;A. baumannii&lt;/i&gt; cells expressing OXA-23 and OXA-24/40 contain these enzymes in their active form. Moreover, OXA-loaded OMVs were able to protect &lt;i&gt;A. baumannii&lt;/i&gt;, &lt;i&gt;Escherichia coli&lt;/i&gt;, and &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; cells susceptible to piperacillin and imipenem. These results permit us to conclude that membrane binding is a bacterial host-specific phenomenon in OXA enzymes. These findings reveal that membrane-bound β-lactamases are more common than expected and support the hypothesis that OMVs loaded with lipidated β-lactamases are vehicles for antimicrobial resistance and its dissemination. This advantage could be crucial in polymicrobial infections, in which &lt;i&gt;Acinetobacter&lt;/i&gt; spp. are usually involved, and underscore the relevance of identifying the cellular localization of lactamases to better understand their physiology and target them.IMPORTANCEβ-lactamases represent the main mechanism of antimicrobial resistance in Gram-negative pathogens. Their catalytic function (cleaving β-lactam antibiotics) occurs in the bacterial periplasm, where they are commonly reported as soluble proteins. A bioinformatic analysis reveals a significant number of putative lipidated β-lactamases, expected to be attached to the outer bacterial membrane. Notably, 60% of Class D OXA β-lactamases (all from &lt;i&gt;Acinetobacter&lt;/i&gt; spp.) are predicted as membrane-anchored proteins. We demonstrate that two clinically relevant carbapenemases, OXA-23 and OXA-24/40, are membrane-bound proteins in &lt;i&gt;A. baumannii&lt;/i&gt;. This cellular localization favors the secretion of these enzymes into outer membrane vesicles that transport them outside the boundaries of the cell. β-lactamase-loaded vesicles can protect populations of antibiotic-susceptible bacteria, enabling them to thrive in the presence of β-lactam antibiotics. The ubiquity of this phenomenon suggests that it may have influenced the dissemination of resistance mediated by &lt;i&gt;Acinetobacter&lt;/i&gt; spp., particularly in polymicrobial infections, being a potent evolutionary adva","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0334324"},"PeriodicalIF":5.1,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142818505","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}