Microbiology spectrum最新文献

{"title":"Novel application of ribonucleoprotein-mediated CRISPR-Cas9 gene editing in plant pathogenic oomycete species.","authors":"Erika N Dort, Nicolas Feau, Richard C Hamelin","doi":"10.1128/spectrum.03012-24","DOIUrl":"10.1128/spectrum.03012-24","url":null,"abstract":"<p><p>CRISPR-Cas9 gene editing has become an important tool for the study of plant pathogens, allowing researchers to functionally characterize specific genes involved in phytopathogenicity, virulence, and fungicide resistance. Protocols for CRISPR-Cas9 gene editing have already been developed for Phytophthoras, an important group of oomycete plant pathogens; however, these efforts have exclusively focused on agricultural pathosystems, with research lacking for forest pathosystems. We sought to develop CRISPR-Cas9 gene editing in two forest pathogenic Phytophthoras, <i>Phytophthora cactorum</i> and <i>P. ramorum</i>, using a plasmid-ribonucleoprotein (RNP) co-transformation approach. Our gene target in both species was the ortholog of <i>PcORP1</i>, which encodes an oxysterol-binding protein that is the target of the fungicide oxathiapiprolin in the agricultural pathogen <i>P. capsici</i>. We delivered liposome complexes, each containing plasmid DNA and CRISPR-Cas9 RNPs, to <i>Phytophthora</i> protoplasts using a polyethylene glycol-mediated transformation protocol. We obtained two <i>ORP1</i> mutants in <i>P. cactorum</i> but were unable to obtain any mutants in <i>P. ramorum</i>. The two <i>P. cactorum</i> mutants exhibited decreased resistance to oxathiapiprolin, as measured by their radial growth relative to wild-type cultures on oxathiapiprolin-supplemented medium. Our results demonstrate the potential for RNP-mediated CRISPR-Cas9 gene editing in <i>P. cactorum</i> and provide a foundation for future optimization of our protocol in other forest pathogenic <i>Phytophthora</i> species.IMPORTANCECRISPR-Cas9 gene editing has become a valuable tool for characterizing the genetics driving virulence and pathogenicity in plant pathogens. CRISPR-Cas9 protocols are now well-established in several <i>Phytophthora</i> species, an oomycete genus with significant economic and ecological impact globally. These protocols, however, have been developed for agricultural <i>Phytophthora</i> pathogens only; CRISPR-Cas9 systems have not yet been developed for any forest pathogenic Phytophthoras. In this study, we sought to establish CRISPR-Cas9 gene editing in two forest <i>Phytophthora</i> pathogens that cause widespread tree mortality: <i>P. cactorum</i> and <i>P. ramorum</i>. We successfully obtained gene mutations in <i>P. cactorum</i> and demonstrated a decrease in fungicide resistance, a trait that could impact the pathogen's ability to cause disease. However, the same protocol did not yield any mutants in <i>P. ramorum</i>. The results of our study will serve as a baseline for the development of CRISPR-Cas9 gene editing in forest Phytophthoras and other oomycetes.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0301224"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11960053/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143516150","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Profilin Pfy1 is critical for cell wall integrity and virulence in <i>Candida albicans</i>.","authors":"Xun Sun, Yueqing Wang, Xiaomin Yang, Xi Xiang, Lili Zou, Xiaowen Liu, Gang Luo, Qi Han","doi":"10.1128/spectrum.02593-24","DOIUrl":"10.1128/spectrum.02593-24","url":null,"abstract":"<p><p>Profilin is a small actin-binding protein that plays an important role in actin polymerization. However, its functions in <i>Candida albicans</i>, the most prevalent fungal pathogen, remain unclear. Here, we report that profilin plays a crucial role in <i>C. albicans</i> morphogenesis and virulence. Deletion of profilin results in abnormal morphogenesis and impaired hyphal development. Furthermore, <i>pfy1Δ/Δ</i> is hypersensitive to cell wall stress and displays thicker cell wall than wild-type cells, indicative of a critical function of Pfy1 in cell wall integrity. In addition, our findings demonstrate that profilin is required for the virulence of <i>C. albicans</i> in a murine model of systemic infection. In conclusion, our work provides a promising target for developing antifungal drugs.IMPORTANCEOur research revealed Pfy1 is not only involved in hyphal development but also essential for pseudohyphal formation in response to DNA damage agents methyl methanesulfonate (MMS) and H₂O_2. The disruption of <i>PFY1</i> resulted in striking morphological defects in both yeast and hyphal forms. Further investigation suggested that profilin plays a role in polarized growth of <i>Candida albicans</i> via binding with Act1, and contributes to cell wall remodeling. Both hyphal growth and cell wall integrity are the important virulence factors of <i>C. albicans</i>. Thus, <i>pfy1Δ/Δ</i> strains significantly reduced mortality rates in mice. These findings suggested that profilin could serve as a target for developing new antifungal drugs possibly for use in combination therapies with caspofungin, for treating invasive candidiasis.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0259324"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11960436/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143483377","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Association of infection-induced antibody levels with risk of subsequent SARS-COV-2 reinfection among healthcare professionals, Rhode Island, 1 March 2020-17 February 2021.","authors":"Jianrong Shi, M Gayle Gabriel, Monica Epperson, Phil A Chan, Jefferson M Jones, Lyle R Petersen, Melissa Briggs Hagen, Natalie J Thornburg, Sharon Saydah, Claire M Midgley","doi":"10.1128/spectrum.02086-24","DOIUrl":"10.1128/spectrum.02086-24","url":null,"abstract":"<p><p>Numerous studies have investigated vaccine-induced correlates of protection (CoP) against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection, but data on infection-induced CoP are limited. Given differences between vaccine- and infection-induced immune responses, in conjunction with low vaccination in many US populations, a better understanding of infection-induced CoP is needed. We used residual sera from a mid-2020 Rhode Island serosurvey of healthcare professionals (HCP) and corresponding state-collected SARS-CoV-2 testing data through February 2021 to generate an analytic cohort of HCP with a first SARS-CoV-2 infection prior to serosurvey blood collection and multiple viral tests after blood collection to assess for reinfection (defined as a positive viral test ≥90 days after their first positive). We tested sera for levels of IgG and IgA targeting ancestral spike (S), receptor-binding domain (RBD), or nucleocapsid (N). We used adjusted Cox proportional hazard ratios to assess the association between categorical antibody level and the risk of subsequent reinfection. Among 170 HCP included in this analysis (median age = 47 years; interquartile range: 35-55 years), 30 were reinfected during the analytic period. Adjusted Cox proportional hazard ratios indicated that higher levels of anti-S or anti-RBD IgG were significantly associated with a lower risk of reinfection. These findings support the use of anti-S or anti-RBD IgG levels as markers of immunologic protection, such as in population serosurveys, or immune-bridging studies in settings of high prevalence of prior infection.  IMPORTANCEThe measurement of antibodies in blood is a relatively simple process and commonly used to estimate overall levels of past infection in populations. But, if someone has antibodies, does this mean that they are protected from being infected again? And are people with higher levels of antibody better protected? There are good data in the literature exploring how antibodies from the coronavirus disease 2019 (COVID-19) vaccination are associated with protection. But, there is still a lot to learn about protection conferred by antibodies that develop after a severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection. In our study, we measure the levels of six different antibody types developed after infection and compare levels to the risk of subsequent infection to better understand which antibody types are best associated with protection. Our data are important for improving studies that use antibodies as proxies for protection, such as population immunity estimates, or those assessing new prevention products.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0208624"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11960437/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492887","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simplifying SARS-CoV-2 wastewater-based surveillance using an automated FDA EUA assay.","authors":"Shivaprasad H Sathyanarayana, Ashlee A Robins, Diana M Toledo, Torrey L Gallagher, Gregory J Tsongalis, Jacqueline A Hubbard, Joel A Lefferts, Isabella W Martin","doi":"10.1128/spectrum.02490-24","DOIUrl":"10.1128/spectrum.02490-24","url":null,"abstract":"<p><p>Wastewater-based surveillance (WBS) can track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in communities. Laboratory methods for this testing involve labor-intensive, multi-step processes. This study assessed the feasibility of performing WBS with an off-label use of an automated commercial SARS-CoV-2 assay that had received Emergency Use Authorization for human diagnostic testing from the United States Food and Drug Administration (FDA EUA). Twenty-four-hour composite samples of primary influent wastewater from seven municipalities in New Hampshire and Vermont were collected between September 2020 and February 2021, and were centrifuged upon receipt. An aliquot of fresh supernatant was immediately tested with the Abbott <i>m</i>2000 RealTi<i>m</i>e SARS-CoV-2 assay (Abbott Molecular, Des Plaines, IL, USA). Corresponding aliquots were then stored at -80°C until they were thawed, polyethylene glycol (PEG) concentrated, and tested by two PCR-based laboratory-developed tests (LDTs). Wastewater samples (103) were tested with successful detection of SARS-CoV-2 viral RNA by all three methods. Bland-Altman analysis showed overall concordant results with a bias of -0.13 and -0.42 log copies/mL detected by the FDA EUA assay compared to the LDTs. Specimen stability assessment demonstrated a decrease of 33.9% measurable viral RNA after three freeze-thaw cycles. SARS-CoV-2 detection in wastewater using an FDA EUA assay on an automated commercial testing platform performed comparably but with more efficient workflow when compared to two LDTs. This sample-to-answer automated method could save time and labor for surveillance testing, but further validation of its ability to quantitate SARS-CoV-2 viral RNA is necessary.IMPORTANCEThis proof-of-principle study evaluates an off-label use of an automated United States Food and Drug Administration (FDA) Emergency Use Authorization (EUA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) human diagnostic assay for wastewater surveillance. Compared to standard, labor-intensive, multi-step methods currently in use for wastewater surveillance testing, an off-label use of an FDA EUA assay on an automated platform offers a sample-to-answer testing requiring less labor and a faster turnaround time.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0249024"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11960137/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fur-regulated urease contributes to the environmental adaptation of <i>Yersinia pseudotuberculosis</i>.","authors":"Junyang Wang, Peishuai Fu, Xinquan He, Yuqi Liu, Yuxin Zuo, Zhiyan Wei, Yao Wang, Yantao Yang, Changfu Li, Xihui Shen, Lingfang Zhu","doi":"10.1128/spectrum.02756-24","DOIUrl":"10.1128/spectrum.02756-24","url":null,"abstract":"<p><p>Urease converts urea into ammonia and carbon dioxide, providing a nitrogen and carbon source for microbial growth and serving as an important mechanism for human bacterial pathogens to survive in acidic conditions, which can be regulated by many factors. As a global regulator, the ferric uptake regulator (Fur) regulates a series of genes and pathways involved in many different cellular processes and the virulence of the enteric bacterium <i>Yersinia pseudotuberculosis</i> (<i>Yptb</i>). However, whether Fur regulates the urease activity in <i>Yptb</i> was still unknown. In this study, we found that urease is positively regulated by Fur in response to manganese ions (Mn²⁺), and this regulation by Fur is mediated by specific recognition of the promoter region of urease in <i>Yptb</i>. Furthermore, urease is induced by Mn²⁺ via Fur under low nutrient conditions. Moreover, we provided evidence that urease plays an important role in acid and osmotic stress resistance, biofilm formation, and virulence of <i>Yptb</i>. Our findings provide insights into understanding the regulatory mechanism and multiple functions of urease in <i>Yptb</i>.IMPORTANCEUrease catalyzes the breakdown of urea into ammonia and carbamate, which are widely distributed among bacterial species and play an important role as an important acid resistance system and virulence factor. In most bacterial species, urease expression is tightly regulated in response to environmental cues such as nitrogen status, pH, growth phase, substrate availability, or transcriptional regulators. In this study, we found that urease from <i>Yptb</i> is positively regulated by Fur in response to Mn²⁺ under low nutrient conditions, which functions to combat acid and osmotic stress and enhance biofilm formation, and plays a crucial role in virulence. Importantly, this is the first demonstration of a direct role for Fur and Mn²⁺ in regulating urease expression in <i>Yptb</i>. This study provides a comprehensive understanding of the regulatory mechanisms and functions of urease from <i>Yptb</i>.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0275624"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11960103/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human bocavirus-1 infection in hospitalized pediatric patients with acute respiratory tract infections.","authors":"Jie Tan, Ziyin Huang, Wenting Tang, Guangbing Liu, Peiqun Li, Jiaqi Wen, Lishai Mo, Chenglan Yan, Zhao Dang, Huiping Huang, Qifei Li, Chunyun Fu","doi":"10.1128/spectrum.02985-24","DOIUrl":"10.1128/spectrum.02985-24","url":null,"abstract":"<p><p>Human bocavirus-1 (HBoV1) is an emerging viral pathogen associated with acute respiratory tract infections (ARTIs) in pediatric populations. This study aimed to evaluate the infection status and clinical characteristics of HBoV1 among hospitalized children suffering from ARTIs. A cohort of 5,021 pediatric patients with respiratory infections was analyzed using targeted next-generation sequencing to identify HBoV1 and other co-existing respiratory pathogens. Results indicated a detection rate of HBoV1 of 8.48%, predominantly among infants under 24 months, with a higher prevalence observed in males. Among the 426 children with HBoV1 detected, there were 17 cases (4%) of single HBoV1 infection, and 409 cases (96%) were co-infected with HBoV1 and other pathogens. The predominant infection pattern was HBoV1-bacteria-virus co-infection (155 cases). A total of 48 other pathogens were detected in children with HBoV1 co-infections, with rhinovirus and human herpesvirus being the most common. The median hospitalization duration for the children with HBoV1 was 8 days, and 16.26% required ICU admission for monitoring. The study offers a comprehensive analysis of the distribution of co-infecting pathogens, clinical features, and outcomes in hospitalized children with HBoV one infection.</p><p><strong>Importance: </strong>There is currently a poor understanding of the characteristics and clinical manifestations of human bocavirus-1 (HBoV1) infection. In the past decade, few studies have thoroughly analyzed co-infecting pathogens, such as viruses, bacteria, and fungi and their patterns in relation to HBoV1. This study utilizes targeted next-generation sequencing (tNGS) technology to identify HBoV1 and other common respiratory pathogens in 5,021 hospitalized children suffering from respiratory infections in the Guangxi region. It offers a detailed analysis of the distribution of co-infecting pathogens, infection patterns, laboratory findings, clinical manifestations, imaging features, complications, and prognoses related to HBoV1 infection in this population. The study highlights the high co-infection rates associated with HBoV1 and offers important insights for diagnosing, treating, and managing children with HBoV1 infection.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0298524"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11960042/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492955","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genomic characteristics and phylogenetic analyses of colonization and infection with carbapenem-resistant <i>Klebsiella pneumoniae</i> in multicenter intensive care units: a cohort study.","authors":"Yi-Le Wu, Wen-Wen Chu, Xiao-Qian Hu, Yi-Yu Lyu, Jie-Hao Tai, Ruo-Jie Li, Kai Huang, Xue Zhao, Wen-Hui Zhang, Xue-Ping Wang, Xiang Yan, Zhou Liu, Xi-Yao Yang, Qiang Zhou, Min Yang","doi":"10.1128/spectrum.01584-24","DOIUrl":"10.1128/spectrum.01584-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Carbapenem-resistant &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; (&lt;i&gt;CRKp&lt;/i&gt;) poses a major threat to global public health. This study aimed to investigate the genomic characteristics and phylogenetic relatedness of colonization and infection with &lt;i&gt;CRKp&lt;/i&gt; among intensive care unit (ICU) patients. A total of 61 &lt;i&gt;CRKp&lt;/i&gt; isolates, including 29 non-ICU-acquired and 32 ICU-acquired strains, were collected by active screening and infection culture from 16 ICUs through a multicenter cohort study. Following whole-genome sequencing (WGS), seven sequence types (STs) were identified, with ST11 (60.7%) being the most dominant, followed by ST15 (27.9%). Most strains (93.4%) carried the &lt;i&gt;bla&lt;/i&gt;KPC-2 gene, while 9.8% and 3.3% of strains carried either the &lt;i&gt;bla&lt;/i&gt;NDM-1 gene or both genes. A high abundance of virulence genes including &lt;i&gt;iutA&lt;/i&gt; (55.7%), &lt;i&gt;rmpA&lt;/i&gt; (18.0%), and &lt;i&gt;rmpA2&lt;/i&gt; (52.5%) were identified, with &lt;i&gt;iutA +rmpA2&lt;/i&gt; (37.7%) being the most common combination. The IncHI1B plasmid was identified in all of the 34 strains carrying the detected virulence genes. Furthermore, results from cgMLST analysis revealed 10 clusters with highly homogeneous &lt;i&gt;CRKp&lt;/i&gt; strains, from which the potential interregional and intrahospital spread of &lt;i&gt;CRKp&lt;/i&gt; were hypothesized. Here, this study reveals the widespread distribution of &lt;i&gt;CRKp&lt;/i&gt; as well as locally specific strains in different regions of Anhui Province. The observed high abundance of virulence genes requires additional attention and continued monitoring. Taken together, these findings highlight the need for coordinated efforts between healthcare facilities and networks to aid &lt;i&gt;CRKp&lt;/i&gt; control strategies and prevent spread. Moreover, we emphasize the importance of WGS in revealing additional insights, thus improving epidemiological studies and transmission control of &lt;i&gt;CRKp&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;Carbapenem-resistant &lt;i&gt;K. pneumoniae&lt;/i&gt; (&lt;i&gt;CRKp&lt;/i&gt;) has spread rapidly to different parts of the world and poses a serious threat to global health. High genetic diversity in &lt;i&gt;CRKp&lt;/i&gt; can introduce complexities in disease treatment and management. Intensive care unit (ICU) patients are more susceptible to acquire &lt;i&gt;CRKp&lt;/i&gt; infections. However, most &lt;i&gt;CRKp&lt;/i&gt; studies have focused on strains isolated from infections, rather than cases of asymptomatic &lt;i&gt;CRKp&lt;/i&gt; colonization. This study analyzed clinical &lt;i&gt;CRKp&lt;/i&gt; isolates from ICU patients as well as isolate cases of active colonization screening. Findings reveal the genetic diversity of &lt;i&gt;CRKp&lt;/i&gt; in different regions of Anhui Province, emphasizing the necessity for a more comprehensive investigation of the genomic characteristics and phylogenetic relatedness of &lt;i&gt;CRKp&lt;/i&gt; in different regions. Data regarding antimicrobial resistance genes, virulence genes, and genetic relatedness will improve the understanding of the potential risk of &lt;i&gt;CRKp&lt;/i&gt; to public health and aid guidance for prevention and control of &lt;","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0158424"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11960118/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143483407","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"New insights into the testicular tropism of porcine reproductive and respiratory syndrome virus.","authors":"Kassandra Durazo-Martínez, Fernando A Osorio, Gustavo Delhon, Jesús Hernández, Hiep L X Vu","doi":"10.1128/spectrum.02964-24","DOIUrl":"10.1128/spectrum.02964-24","url":null,"abstract":"<p><p>Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted host specificity, primarily infecting porcine macrophages. Notably, an exception to such macrophage-restricted tropism has been observed in sexually active boars, where the virus infects and induces apoptosis in the germinal epithelium, resulting in viral dissemination in the ejaculate. Whether this phenomenon occurs in prepubertal animals remains unclear. In this study, we isolated spermatogonia stem cells (SSCs) from neonatal pigs and cultured them <i>in vitro</i>. These SSC cultures formed morula-like colonies, exhibited alkaline phosphatase activity-a characteristic of stem cells-and expressed protein gene product 9.5, a marker of SSCs. Notably, the SSC cultures supported PRRSV replication with kinetics similar to that observed in porcine alveolar macrophages. To assess the testicular tropism of PRRSV in prepuberal animals, 28-day-old male pigs were infected with a virulent PRRSV strain. Testicular tissues were sequentially analyzed using a combination of <i>in situ</i> hybridization for PRRSV RNA and immunohistochemistry for specific cellular markers. Unlike in sexually active boars, PRRSV did not infect the spermatogonia cells within the seminiferous tubules of prepubertal pigs. Instead, the virus primarily infected macrophages and myoid cells located in the interstitium and peritubular areas. It appeared that the anatomical separation of spermatogonia from the basal membrane of the seminiferous tubules in prepubertal pigs prevents these cells from being infected by PRRSV. Overall, our findings offer valuable insights into the age-dependent testicular tropism of PRRSV.IMPORTANCEContaminated boar semen used in artificial insemination has significantly contributed to the global spread of porcine reproductive and respiratory syndrome virus (PRRSV), a virus that typically infects only cells within the monocyte and macrophage lineages. Our study reveals that spermatogonia stem cells (SSCs) from neonatal piglets are also susceptible to PRRSV, suggesting that non-macrophage cells can be infected by the virus. However, despite this susceptibility, PRRSV-infected cells were not found in the seminiferous tubules of prepubertal male pigs inoculated with a virulent PRRSV strain. This contrasts with sexually mature boars, where PRRSV-infected cells were prominently observed within the seminiferous tubules. The discrepancy is likely due to anatomical differences between the seminiferous tubules of sexually mature boars and prepubertal pigs. These findings provide new insights into PRRSV pathogenesis. Additionally, the <i>ex vivo</i> SSC culture provides a valuable model for identifying new viral receptors necessary for PRRSV infection and for investigating the virus's impact on spermatogenesis.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0296424"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11960452/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449550","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Targeting efflux pumps prevents the multi-step evolution of high-level resistance to fluoroquinolone in <i>Pseudomonas aeruginosa</i>.","authors":"Xiao-Quan Yu, Hao Yang, Han-Zhong Feng, Jun Hou, Jun-Qiang Tian, Shao-Min Niu, Chong-Ge You, Xuan-Yu Tao, Si-Ping Zhang, Zhi-Ping Wang, Yong-Xing He","doi":"10.1128/spectrum.02981-24","DOIUrl":"10.1128/spectrum.02981-24","url":null,"abstract":"<p><p>Antibiotic resistance is emerging as a significant global health crisis, necessitating the urgent development of novel antibiotics or alternative therapies. Although it is recognized that bacteria require multiple mutations to develop resistance levels exceeding the mutant prevention concentration, the specific mutation combinations conferring high resistance have been largely undefined. Here, we investigated the multi-step evolution of fluoroquinolone resistance in <i>Pseudomonas aeruginosa</i> through experimental evolution and whole-genome sequencing coupled with proteomic approaches. We discovered that in low-dose and high-dose experimental evolution scenarios, combinations of mutations in the negative regulators of efflux pumps (<i>nfxB/mexR</i>) and DNA gyrases (<i>gyrA/gyrB</i>) contributed to the high-level resistance and some of these combinations were also prevalent in clinical isolates of <i>P. aeruginosa</i>. Notably, the selected <i>nfxB</i> mutation, which resulted in the overexpression of the MexCD-OprJ efflux pump, also exhibited collateral sensitivity to aminoglycosides and enhanced antibiotic tolerance. It was further revealed that the efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) could effectively prevent evolution to high-level resistance for both laboratory and clinical <i>P. aeruginosa</i> strains. Our work highlights the critical role of efflux pump repressor-related mutations in the evolution of high-level antibiotic resistance and demonstrates the potential of targeting these mutations to impede the evolution toward high-level resistance.IMPORTANCEIn this study, we examined the stepwise evolution of fluoroquinolone resistance in <i>Pseudomonas aeruginosa</i> using experimental evolution, whole-genome sequencing, and proteomic analyses. Our findings revealed that under both low-dose and high-dose conditions, mutations in efflux pump regulators (<i>nfxB</i>/<i>mexR</i>) and DNA gyrase genes (<i>gyrA</i>/<i>gyrB</i>) synergistically contributed to high-level resistance. These mutation combinations were not only observed in experimental settings but also detected in clinical isolates of <i>P. aeruginosa</i>. This work underscores the pivotal role of efflux pump repressor-related mutations in the progression to high-level antibiotic resistance. It also highlights the promise of targeting efflux pumps as a strategy to prevent the multi-step evolution of resistance in <i>P. aeruginosa</i>.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0298124"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11960432/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143468399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Marek's disease virus protein kinase US3 inhibits DNA-sensing antiviral innate immunity via abrogating activation of NF-κB.","authors":"Kai Li, Rui Liu, Yongzhen Liu, Li Gao, Changjun Liu, Yanping Zhang, Xiaole Qi, Hongyu Cui, Suyan Wang, Yuntong Chen, Yulu Duan, Yulong Gao, Xiaomei Wang","doi":"10.1128/spectrum.02347-24","DOIUrl":"10.1128/spectrum.02347-24","url":null,"abstract":"<p><p>Marek's disease virus (MDV) is an avian alphaherpesvirus associated with Marek's disease, an immunosuppressive and lymphoproliferative disease in chickens. The DNA sensing pathway mediates innate immune defense against infection by many DNA-containing pathogens, while viruses have evolved multiple strategies to evade the host immune response to survive in host cells. This study found that ectopic expression of MDV protein kinase US3 inhibited beta interferon (IFN-β) and interleukin-6 (IL-6) production induced by interferon-stimulatory and viral DNA. US3 was further shown to abolish the nuclear factor κB (NF-κB) activation. The US3 kinase activity was indispensable for its inhibitory function, as the kinase-dead US3 mutant (US3K220A) did not inhibit NF-κB activation. Further studies showed that US3 interacted with the Rel homology domains of the NF-κB subunits p65 and p50, which phosphorylated these transcription factors and blocked their nuclear translocation. Finally, US3 deficiency promoted IFN-β and IL-6 production, resulting in reduced viral replication and lower MDV-specific lesion incidence during MDV infection in chickens. Altogether, these findings reveal a novel mechanism for MDV to evade host antiviral immunity.IMPORTANCEMarek's disease virus (MDV) is an oncogenic avian alphaherpesvirus that causes an economically important disease affecting the health and welfare of poultry worldwide. Whereas human herpesviruses have been shown to evolve various strategies to inhibit the DNA sensing signaling for the evasion of the host's innate immunity, little is known regarding the mechanism for MDV to regulate this pathway. In this study, MDV US3 protein kinase was demonstrated to inhibit the activation of NF-κB in the DNA sensing pathway via binding to the Rel homology domains of the NF-κB subunits p65 and p50, which hyperphosphorylated these transcription factors and abolished their nuclear translocation. This is an important finding toward a better understanding of the functions of avian alphaherpesviruses encoded US3 protein kinase.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0234724"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11960123/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143557177","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}