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Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria and, in doing so, may promote the spread of drug resistance genes in the population.IMPORTANCEThis work extends our understanding of horizontal gene transfer and the roles of extracellular vesicles in pneumococcus. This bacterium serves as the model for transformation, a process by which bacteria can take up naked DNA from the environment. Here, we show that extracellular vesicles secreted by the pneumococcus have DNA on their surface and that this DNA can be imported by the transformation machinery, facilitating gene transfer. Understanding EV-mediated gene transfer may provide new avenues to manage the spread of antibiotic drug resistance.

Archaea catalyzing the first step of nitrification in the rhizosphere possibly have an influence on plant growth and development. In this study, we found a distinct archaeal community, dominated by ammonia-oxidizing archaea (AOA), associated with the root system of pepper (Capsicum anuum L.) and ginseng plants (Panax ginseng C.A. Mey.) compared to bulk soil not penetrated by roots. While the abundance of total AOA decreased in the rhizosphere soils, AOA related to "Candidatus Nitrosocosmicus," which harbor gene encoding manganese catalase (MnKat) in contrast to most other AOA, dominated the AOA community in the rhizosphere soils. For both plant species, the ratio of copy numbers of the AOA MnKat gene to the amoA gene (encoding the ammonia monooxygenase subunit A) was significantly higher in the rhizospheres than in bulk soils. In contrast to MnKat-negative strains from other AOA clades, the catalase activity of a representative isolate of "Ca. Nitrosocosmicus" was demonstrated. Members of this clade were enriched in H₂O₂-amended bulk soils, and constitutive expression of their MnKat gene was observed in both bulk and rhizosphere soils. Due to their abundance, "Ca. Nitrosocosmicus" members can be considered important players mediating the nitrification process in rhizospheres. The dominance of this MnKat-containing AOA in rhizospheres of agriculturally important plants hints at a previously overlooked AOA-plant interaction.

Importance: Ammonia-oxidizing archaea (AOA) are widespread in terrestrial environments and outnumber other ammonia oxidizers in the rhizosphere, possibly exerting an influence on plant growth and development. However, little is known about the selection forces that shape their composition, functions, survival, and proliferation strategies in the rhizosphere. Here, we observed a distinct AOA community on root systems of two different plant species compared to bulk soil. Our results show that the "Ca. Nitrosocosmicus" clade, which possesses functional MnKat genes unlike most other AOA, dominated the rhizosphere soils. Moreover, members of this clade were enriched in H2O2-amended bulk soil, which mimics the ROS stress in root systems. While research on AOA-plant interactions in the rhizosphere is still in its infancy, these findings suggest that "Ca. Nitrosocosmicus" may be an important clade of AOA with potential AOA-plant interaction.

Previous studies in Ghana indicated low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and predominance of ST152 methicillin-susceptible S. aureus (MSSA) among clinical isolates. ST152 MRSA clones are associated with severe infections and epidemics. Using whole genome sequencing (WGS), 159 S. aureus isolated from clinical sources (wound, blood, urine, ear, abscess, umbilical cord, eye, vaginal samples, and others) from 10 hospitals across Ghana were investigated. mecA (gene for methicillin resistance) was detected in 38% of the isolates. Panton-Valentine leucocidin toxin (PVL) gene occurred in 65% isolates, with 84% of the MRSA's harboring the PVL gene. ST152 was the major clone, with 74% harboring the mecA gene. Other MRSA clones detected were ST5, ST5204, ST852, and ST1. MSSA clones included ST3249, ST152, ST5, ST1, and ST8. Twenty-three genes encoding resistance to 12 antimicrobial classes were observed with blaZ (97%) being the most prevalent. Other predominant resistance genes included tetK (46%), cat (42%), and dfrG (36%) encoding resistance for tetracyclines, phenicols, and diaminopyrimidine, respectively. Virulence genes for enterotoxins, biofilms, toxic-shock-syndrome toxins, hemolysins, and leukotoxins were also detected. Phylogenetic analysis revealed a shift in the dominant clone from MSSA ST152 to MRSA ST152 over the past decade. The study provides valuable insights into the genomic content of S. aureus from clinical sources in Ghana. The finding of ST152 MRSA in high numbers suggests a shifting epidemiological landscape of these pathogens and continuous surveillance using robust tools like WGS is needed to monitor the rise and spread of these epidemic clones in the country.IMPORTANCESince its emergence in 1959, MRSA has been a significant public health concern, causing infections in both clinical and community settings. Patients with MRSA-related infections experience higher mortality rates due to its ability to evade antimicrobials and immune defenses. In Ghana, understanding the molecular epidemiology of MRSA has been hindered by the lack of appropriate laboratory infrastructure and the limited capacity for molecular data analysis. This study, the largest genomic study of S. aureus in Ghana, addresses this gap by utilizing whole genome sequencing to examine the diversity of circulating S. aureus strains from 10 hospitals. Our findings highlight the predominance of pandemic clones, particularly ST152, and the notable transition of ST152 MSSA to ST152 MRSA over the past decade. The findings from this study supports AMR surveillance efforts in Ghana and emphasize the importance of implementing genomic surveillance using WGS to comprehensively monitor the rise and spread of multi-drug-resitant organisms such as MRSA in the country.

The plasma membrane is critical for the virulence of the human fungal pathogen Candida albicans. In addition to functioning as a protective barrier, the plasma membrane plays dynamic roles in a wide range of functions needed for virulence including nutrient uptake, cell wall synthesis, morphogenesis, resistance to stress, and invasive hyphal growth. Screening a collection of C. albicans mutants identified an understudied gene that is important for invasive hyphal growth, which we have termed CWR1 (Cell Wall Regulatory kinase). A mutant strain lacking CWR1 displayed defects in resisting stressful conditions that exacerbate cell wall defects. The Cwr1 protein shows strong similarity to protein kinases, suggesting it plays a regulatory role in coordinating plasma membrane and cell wall functions. A Cwr1-green fluorescent protein (GFP) fusion protein localized to punctate patches associated with the plasma membrane that partially overlapped Membrane Compartment of Can1 (MCC)/eisosome domains. In contrast to the static MCC/eisosome domains, the Cwr1-GFP patches were very dynamic. Truncation mutants lacking C-terminal sequences distal to the protein kinase domain failed to show detectable localization at the plasma membrane. Surprisingly, these mutant strains did not show the defects of a cwr1Δ mutant, suggesting that localization to punctate patches associated with the plasma membrane is not essential for Cwr1 function. Altogether, these data indicate that Cwr1 contributes to the regulation of plasma membrane functions that promote proper morphogenesis and resistance to cell wall stress, both of which are important for C. albicans virulence.

Importance: The ability of Candida albicans to grow invasively in the host and resist stress is critical for it to be an effective human pathogen. Identifying the genes that promote these processes is important for developing new strategies to block infection. Therefore, genetic methods were used in this study to identify a novel gene that is needed for invasive growth and stress resistance (Cell Wall Regulatory kinase [CWR1]). Interestingly, the Cwr1 protein localized to punctate patches in the plasma membrane, some of which co-localized with specialized subdomains of the plasma membrane known as eisosomes that are known to promote stress resistance and invasive growth in the host. Thus, these studies identified a novel regulator of traits that are critical for C. albicans pathogenesis.

Dr. Parimal Samir works in the field of host-pathogen interactions. In this mSphere of Influence article, he reflects on how the manuscript entitled "De novo gene synthesis by an antiviral reverse transcriptase" by Samuel Sternberg and colleagues made an impact by reminding him that there is still so much to discover in life sciences.

Host cell damage is a key parameter for research in infection biology, drug testing, and substance safety screening. In this study, we introduce a luciferase reporter system as a new and reliable assay to measure cell damage and validate it with the pathogenic yeast, Candida albicans, as a test case. We transduced human epithelial cell lines with a lentiviral vector to stably express an optimized luciferase enzyme, Nanoluc. Upon cell damage, the release of cytoplasmic luciferase into the extracellular space can be easily detected by a luminometer. We used the luciferase reporter system to investigate the damage caused by C. albicans to different newly generated epithelial reporter cell lines. We found that fungus-induced cell damage, as determined by established methods, correlated tightly with the release of the luciferase. The new luciferase reporter system is a simple, sensitive, robust, and inexpensive method for measuring host cell damage and has a sensitivity comparable to the standard assay, release of lactate dehydrogenase. It is suitable for high-throughput studies of pathogenesis mechanisms of any microbe, for antimicrobial drug screening, and many other applications.IMPORTANCEWe present a quick, easy, inexpensive, and reliable assay to measure damage to mammalian cells. To this end, we created reporter cell lines which artificially express luciferase, an enzyme that can be easily detected in the supernatant when these cells are damaged. We used infections with the well-investigated fungal pathogen of humans, Candida albicans, as a test case of our system. Using our reporter, we were able to recapitulate the known effects of strain variability, gene deletions, and antifungal treatments on host cell damage. This easily adaptable reporter system can be used to screen for damage in infection models with different microbial species, assay cell-damaging potential of substances, discover new non-toxic antibiotics, and many other damage-based applications.

The obligate intracellular pathogen, Chlamydia trachomatis, establishes an intracellular niche within a host membrane-derived vacuole called the chlamydial inclusion. From within this inclusion, C. trachomatis orchestrates numerous host-pathogen interactions, in part, by utilizing a family of type III secreted effectors, termed inclusion membrane proteins (Incs). Incs are embedded within the inclusion membrane, and some function to recruit host proteins to the inclusion. Two such recruited host proteins are leucine rich repeat Flightless-1 interacting protein 1 (LRRF1/LRRFIP1) and its binding partner Flightless 1 (FLI1/FLII). Previously, LRRF1 has been shown to interact with Inc protein Ct226/CTL0478. This is the first study to examine interactions of FLI1 with candidate Incs or with LRRF1 during infection. We hypothesized that FLI1 recruitment to the inclusion would be dependent on LRRF1 localization. We demonstrated that FLI1 co-immunoprecipitated with Ct226 but only in the presence of LRRF1. Furthermore, FLI1 localized to the inclusion when LRRF1 was depleted via small interfering RNA, suggesting that FLI1 may have an alternative recruitment mechanism. We further developed a series of CRISPRi knockdown and complementation strains in C. trachomatis serovar L2 targeting ct226 and co-transcribed candidate Incs, ct225 and ct224. Simultaneous knockdown of ct226, ct225, and ct224 prevented localization of both FLI1 and LRRF1 to the inclusion, and only complementation of ct226 restored their localization. Thus, we demonstrated Ct226 is critical for FLI1 and LRRF1 localization to the inclusion. Our results also indicate an LRRF1-independent localization mechanism for FLI1, which likely influence their mechanism(s) of action during chlamydial infection.IMPORTANCEChlamydia trachomatis is a leading cause of both bacterial sexually transmitted infections and preventable infectious blindness worldwide. As an obligate intracellular pathogen, C. trachomatis has evolved multiple ways of manipulating the host to establish a successful infection. As such, it is important to understand host-chlamydial protein-protein interactions as these reveal strategies that C. trachomatis uses to shape its intracellular environment. This study looks in detail at interactions of two host proteins, FLI1 and LRRF1, during chlamydial infection. Importantly, the series of CRISPR inference knockdown and complement strains developed in this study suggest these proteins have both independent and overlapping mechanisms for localization, which ultimately will dictate how these proteins function during chlamydial infection.