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The endosymbiont increases resistance to pathogens by enhancing iron sequestration and melanization.MBio Jun 2024Facultative endosymbiotic bacteria, such as and species, are commonly found in association with insects and can dramatically alter their host physiology. Many...
UNLABELLED
Facultative endosymbiotic bacteria, such as and species, are commonly found in association with insects and can dramatically alter their host physiology. Many endosymbionts are defensive and protect their hosts against parasites or pathogens. Despite the widespread nature of defensive insect symbioses and their importance for the ecology and evolution of insects, the mechanisms of symbiont-mediated host protection remain poorly characterized. Here, we utilized the fruit fly and its facultative endosymbiont to characterize the mechanisms underlying symbiont-mediated host protection against bacterial and fungal pathogens. Our results indicate a variable effect of on infection outcome, with endosymbiont-harboring flies being more resistant to , and but more sensitive or as sensitive as endosymbiont-free flies to the infections with species. Further focusing on the protective effect, we identified Transferrin-mediated iron sequestration induced by as being crucial for the defense against and . In the case of , enhanced melanization in -harboring flies plays a major role in protection. Both iron sequestration and melanization induced by require the host immune sensor protease Persephone, suggesting a role of proteases secreted by the symbiont in the activation of host defense reactions. Hence, our work reveals a broader defensive range of than previously appreciated and adds nutritional immunity and melanization to the defensive arsenal of symbionts.
IMPORTANCE
Defensive endosymbiotic bacteria conferring protection to their hosts against parasites and pathogens are widespread in insect populations. However, the mechanisms by which most symbionts confer protection are not fully understood. Here, we studied the mechanisms of protection against bacterial and fungal pathogens mediated by the endosymbiont . We demonstrate that besides the previously described protection against wasps and nematodes, also confers increased resistance to pathogenic bacteria and fungi. We identified -induced iron sequestration and melanization as key defense mechanisms. Our work broadens the known defense spectrum of and reveals a previously unappreciated role of melanization and iron sequestration in endosymbiont-mediated host protection. We propose that the mechanisms we have identified here may be of broader significance and could apply to other endosymbionts, particularly to , and potentially explain their protective properties.
PubMed: 38940615
DOI: 10.1128/mbio.00936-24 -
MSystems Jun 2024Over almost three decades, average nucleotide identity (ANI) analysis has been instrumental in operationally defining species in bacteria. However, barely any attention...
Over almost three decades, average nucleotide identity (ANI) analysis has been instrumental in operationally defining species in bacteria. However, barely any attention has been paid to soundly defining intra-species units employing ANI analyses until recently. Notably, some very recent publications are good steps forward in that direction. The level of granularity provided by these intra-species units will be relevant to understanding the eco-evolutionary dynamics and transmission of bacterial lineages and mobile genetic elements, antibiotic resistance, and virulence genes. These intra-species units will undoubtedly advance the genomic epidemiology of many bacterial pathogens. In the coming years, we anticipate that many studies will implement ANI-based definitions of different intra-species units, such as strains or sequence types, for many different bacterial species.
PubMed: 38940600
DOI: 10.1128/msystems.00584-24 -
Microbiology Spectrum Jun 2024The hospital environmental microbiome, which can affect patients' and healthcare workers' health, is highly variable and the drivers of this variability are not well...
UNLABELLED
The hospital environmental microbiome, which can affect patients' and healthcare workers' health, is highly variable and the drivers of this variability are not well understood. In this study, we collected 37 surface samples from the neonatal intensive care unit (NICU) in an inpatient hospital before and after the operation began. Additionally, healthcare workers collected 160 surface samples from five additional areas of the hospital. All samples were analyzed using 16S rRNA gene amplicon sequencing, and the samples collected by healthcare workers were cultured. The NICU samples exhibited similar alpha and beta diversities before and after opening, which indicated that the microbiome there was stable over time. Conversely, the diversities of samples taken after opening varied widely by area. Principal coordinate analysis (PCoA) showed the samples clustered into two distinct groups: high alpha diversity [the pediatric intensive care unit (PICU), pathology lab, and microbiology lab] and low alpha diversity [the NICU, pediatric surgery ward, and infection prevention and control (IPAC) office]. Least absolute shrinkage and selection operator (LASSO) classification models identified 156 informative amplicon sequence variants (ASVs) for predicting the sample's area of origin. The testing accuracy ranged from 86.37% to 100%, which outperformed linear and radial support vector machine (SVM) and random forest models. ASVs of genera that contain emerging pathogens were identified in these models. Culture experiments had identified viable species among the samples, including potential antibiotic-resistant bacteria. Though area type differences were not noted in the culture data, the prevalences and relative abundances of genera detected positively correlated with 16S sequencing data. This study brings to light the microbial community temporal and spatial variation within the hospital and the importance of pathogenic and commensal bacteria to understanding dispersal patterns for infection control.
IMPORTANCE
We sampled surface samples from a newly built inpatient hospital in multiple areas, including areas accessed by only healthcare workers. Our analysis of the neonatal intensive care unit (NICU) showed that the microbiome was stable before and after the operation began, possibly due to access restrictions. Of the high-touch samples taken after opening, areas with high diversity had more potential external seeds (long-term patients and clinical samples), and areas with low diversity and had fewer (short-term or newborn patients). Classification models performed at high accuracy and identified biomarkers that could be used for more targeted surveillance and infection control. Though culturing data yielded viability and antibiotic-resistance information, it disproportionately detected the presence of genera relative to 16S data. This difference reinforces the utility of 16S sequencing in profiling hospital microbiomes. By examining the microbiome over time and in multiple areas, we identified potential drivers of the microbial variation within a hospital.
PubMed: 38940596
DOI: 10.1128/spectrum.00296-24 -
Microbiology Spectrum Jun 2024Traditionally, successful vaccines rely on specific adaptive immunity by activating lymphocytes with an attenuated pathogen, or pathogen subunit, to elicit heightened...
UNLABELLED
Traditionally, successful vaccines rely on specific adaptive immunity by activating lymphocytes with an attenuated pathogen, or pathogen subunit, to elicit heightened responses upon subsequent exposures. However, recent work with and other pathogens has identified a role for "trained" monocytes in protection through memory-like but non-specific immunity. Here, we used an co-culture approach to study the potential role of trained macrophages, including lung alveolar macrophages, in immune responses to the Live Vaccine Strain (LVS) of is an intracellular bacterium that replicates within mammalian macrophages and causes respiratory as well as systemic disease. We vaccinated mice with LVS and then obtained lung alveolar macrophages, or derived macrophages from bone marrow. LVS infected and replicated comparably in both types of macrophages, whether naïve or from LVS-vaccinated mice. LVS-infected macrophages were then co-cultured with either naïve splenocytes, splenocytes from mice vaccinated intradermally, or splenocytes from mice vaccinated intravenously. For the first time, we show that immune (but not naïve) splenocytes controlled bacterial replication within alveolar macrophages, similar to previous results using bone marrow-derived macrophage. However, no differences in control of intramacrophage bacterial replication were found between co-cultures with naïve macrophages or macrophages from LVS-vaccinated mice; furthermore, nitric oxide levels and interferon-gamma production in supernatants were largely comparable across all conditions. Thus, in the context of co-cultures, the data do not support development of trained macrophages in bone marrow or lungs of mice vaccinated with LVS intradermally or intravenously.
IMPORTANCE
The discovery of non-specific "trained immunity" in monocytes has generated substantial excitement. However, to date, training has been studied with relatively few microbes (e.g., Bacille Calmette-Guérin, a live attenuated intracellular bacterium used as a vaccine) and microbial substances (e.g., LPS), and it remains unclear whether training during infection is common. We previously demonstrated that vaccination of mice with Live Vaccine Strain (LVS), another live attenuated intracellular bacterium, protected against challenge with the unrelated bacterium . The present study therefore tested whether LVS vaccination engenders trained macrophages that contributed to this protection. To do so, we used a previous co-culture approach with murine bone marrow-derived macrophages to expand and study lung alveolar macrophages. We demonstrated that alveolar macrophages can be productively infected and employed to characterize interactions with LVS-immune lymphocytes. However, we find no evidence that either bone marrow-derived or alveolar macrophages are trained by LVS vaccination.
PubMed: 38940590
DOI: 10.1128/spectrum.00028-24 -
MBio Jun 2024Human adenoviruses (HAdVs) are small DNA viruses that generally cause mild disease. Certain strains, particularly those belonging to species B HAdVs, can cause severe...
Human adenoviruses (HAdVs) are small DNA viruses that generally cause mild disease. Certain strains, particularly those belonging to species B HAdVs, can cause severe pneumonia and have a relatively high mortality rate. Little is known about the molecular aspects of how these highly pathogenic species affect the infected cell and how they suppress innate immunity. The present study provides molecular insights into how species B adenoviruses suppress the interferon signaling pathway. Our study shows that these viruses, unlike HAdV-C2, are resistant to type I interferon. This resistance likely arises due to the highly efficient suppression of interferon-stimulated gene expression. Unlike in HAdV-C2, HAdV-B7 and B14 sequester STAT2 and RNA polymerase II from interferon-stimulated gene promoters in infected cells. This results in suppressed interferon- stimulated gene activation. In addition, we show that RuvBL1 and RuvBL2, cofactors important for RNA polymerase II recruitment to promoters and interferon-stimulated gene activation, are redirected to the cytoplasm forming high molecular weight complexes that, likely, are unable to associate with chromatin. Proteomic analysis also identified key differences in the way these viruses affect the host cell, providing insights into species B-associated high pathogenicity. Curiously, we observed that at the level of protein expression changes to the infected cell, HAdV-C2 and B7 were more similar than those of the same species, B7 and B14. Collectively, our study represents the first such study of innate immune suppression by the highly pathogenic HAdV-B7 and B14, laying an important foundation for future investigations.IMPORTANCEHuman adenoviruses form a large family of double-stranded DNA viruses known for a variety of usually mild diseases. Certain strains of human adenovirus cause severe pneumonia leading to much higher mortality and morbidity than most other strains. The reasons for this enhanced pathogenicity are unknown. Our study provides a molecular investigation of how these highly pathogenic strains might inactivate the interferon signaling pathway, highlighting the lack of sensitivity of these viruses to type I interferon in general while providing a global picture of how viral changes in cellular proteins drive worse disease outcomes.
PubMed: 38940561
DOI: 10.1128/mbio.01038-24 -
MBio Jun 2024Autophagy is an important biological process in host defense against viral infection. However, many viruses have evolved various strategies to disrupt the host antiviral...
UNLABELLED
Autophagy is an important biological process in host defense against viral infection. However, many viruses have evolved various strategies to disrupt the host antiviral system. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus with a large economic impact on the swine industry. At present, studies on the escape mechanism of PRRSV in the autophagy process, especially through chaperone-mediated autophagy (CMA), are limited. This study confirmed that PRRSV glycoprotein 5 (GP5) could disrupt the formation of the GFAP-LAMP2A complex by inhibiting the MTORC2/PHLPP1/GFAP pathway, promoting the dissociation of the pGFAP-EF1α complex, and blocking the K63-linked polyubiquitination of LAMP2A to inhibit the activity of CMA. Further research demonstrated that CMA plays an anti-PRRSV role by antagonizing nonstructural protein 11 (NSP11)-mediated inhibition of type I interferon (IFN-I) signaling. Taken together, these results indicate that PRRSV GP5 inhibits the antiviral effect of CMA by targeting LAMP2A. This research provides new insight into the escape mechanism of immunosuppressive viruses in CMA.
IMPORTANCE
Viruses have evolved sophisticated mechanisms to manipulate autophagy to evade degradation and immune responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus that causes enormous economic losses in the swine industry. However, the mechanism by which PRRSV manipulates autophagy to defend against host antiviral effects remains unclear. In this study, we found that PRRSV GP5 interacts with LAMP2A and disrupts the formation of the GFAP-LAMP2A complex, thus inhibiting the activity of CMA and subsequently enhancing the inhibitory effect of the NSP11-mediated IFN-I signaling pathway, ultimately facilitating PRRSV replication. Our study revealed a novel mechanism by which PRRSV escapes host antiviral effects through CMA, providing a potential host target, LAMP2A, for developing antiviral drugs and contributing to understanding the escape mechanism of immunosuppressive viruses.
PubMed: 38940560
DOI: 10.1128/mbio.00532-24 -
MBio Jun 2024Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA...
Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has three predicted extracellular hydrolase domains: metallo-peptidase (LytM), soluble lytic transglycosylase (SLT), and cysteine, histidine-dependent amidohydrolases/peptidases (CHAP). Here, we report the structure of the LytM domain and show that its active site is degenerate and lacks the active site metal. Furthermore, we show that only the predicted SLT domain is functional and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Furthermore, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information for understanding the function of Gram-positive T4SSs.IMPORTANCEAntibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from . This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in as well as other medically relevant Gram-positive bacteria.
PubMed: 38940556
DOI: 10.1128/mbio.00488-24 -
MBio Jun 2024Transposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seq is...
UNLABELLED
Transposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seq is challenging. Dose limitations and host restrictions create bottlenecks that diminish the transposon mutant pool being screened. Here, we have developed a murine model with a disruption in that renders the resulting RECON mouse resistant to high-dose infection. We leveraged this model to perform a Tn-seq screen of the human pathogen . We identified 135 genes which were required for growth in mice including novel genes not previously identified for host survival. We identified organ-specific requirements for survival and investigated the role of the folate enzyme FolD in liver pathogenesis. A mutant lacking was impaired for growth in murine livers by 2.5-log compared to wild type and failed to spread cell-to-cell in fibroblasts. In contrast, a mutant in which encodes a transcription factor that represses an operon involved in D-allose catabolism, was attenuated in both livers and spleens of mice by 4-log and 3-log, respectively, but showed modest phenotypes in models. We confirmed that dysregulation of the D-allose catabolism operon is responsible for the growth defect, as deletion of the operon in the ∆ background rescued virulence. By undertaking an unbiased, genome-wide screen in mice, we have identified novel fitness determinants for host infection, which highlights the utility of the RECON mouse model for future screening efforts.
IMPORTANCE
is the gram-positive bacterium responsible for the food-borne disease listeriosis. Although infections with are limiting in healthy hosts, vulnerable populations, including pregnant and elderly people, can experience high rates of mortality. Thus, understanding the breadth of genetic requirements for survival will present new opportunities for treatment and prevention of listeriosis. We developed a murine model of infection using a RECON mouse that is restrictive to systemic infection. We utilized this model to screen for genes required via transposon sequencing. We identified the liver-specific gene and a repressor, , that only exhibits an growth defect. AlsR controls the expression of the D-allose operon which is a marker in diagnostic techniques to identify pathogenic . A better understanding of the role of the D-allose operon in human disease may further inform diagnostic and prevention measures.
PubMed: 38940553
DOI: 10.1128/mbio.01332-24 -
Microbiology Spectrum Jun 2024The majority of the nearly 10,000 described species of green algae are photoautotrophs; however, some species have lost their ability to photosynthesize and become...
UNLABELLED
The majority of the nearly 10,000 described species of green algae are photoautotrophs; however, some species have lost their ability to photosynthesize and become obligate heterotrophs that rely on parasitism for survival. Two high-quality genomes of the heterotrophic algae Pz20 and Pz23 were obtained using short- and long-read genomic as well as transcriptomic data. The genome sizes were 31.2 Mb and 31.3 Mb, respectively, and contig N50 values of 1.99 Mb and 1.26 Mb. Although maintained its plastid genome, the transition to heterotrophy led to a reduction in both plastid and nuclear genome size, including the loss of photosynthesis-related genes from both the nuclear and plastid genomes and the elimination of genes encoding for carotenoid oxygenase and pheophorbide an oxygenase. The loss of genes, including basic leucine-zipper (bZIP) transcription factors, flavin adenine dinucleotide-linked oxidase, and helicase, could have played a role in the transmission of autotrophy to heterotrophs and in the processes of abiotic stress resistance and pathogenicity. A total of 66 (1.37%) and 73 (1.49%) genes were identified as potential horizontal gene transfer events in the two genomes, respectively. Genes for malate synthase and isocitrate lyase, which are horizontally transferred from bacteria, may play a pivotal role in carbon and nitrogen metabolism as well as the pathogenicity of and non-photosynthetic organisms. The two high-quality genomes provide new insights into their evolution as obligate heterotrophs and pathogenicity.
IMPORTANCE
The genus , characterized by its heterotrophic nature and pathogenicity, serves as an exemplary model for investigating pathobiology. The limited understanding of the protothecosis infectious disease is attributed to the lack of genomic resources. Using HiFi long-read sequencing, both nuclear and plastid genomes were generated for two strains of . The findings revealed a concurrent reduction in both plastid and nuclear genome size, accompanied by the loss of genes associated with photosynthesis, carotenoid oxygenase, basic leucine-zipper (bZIP) transcription factors, and others. The analysis of horizontal gene transfer revealed the presence of 1.37% and 1.49% bacterial genes, including malate synthase and isocitrate lyase, which play crucial roles in carbon and nitrogen metabolism, as well as pathogenicity and obligate heterotrophy. The two high-quality genomes represent valuable resources for investigating their adaptation and evolution as obligate heterotrophs, as well as for developing future prevention and treatment strategies against protothecosis.
PubMed: 38940543
DOI: 10.1128/spectrum.04148-23 -
MSystems Jun 2024Skin ulceration syndrome (SUS) is currently the main disease threatening aquaculture due to its higher mortality rate and infectivity, which is caused by . Our previous...
Skin ulceration syndrome (SUS) is currently the main disease threatening aquaculture due to its higher mortality rate and infectivity, which is caused by . Our previous studies have demonstrated that SUS is accompanied by intestinal microbiota (IM) dysbiosis, alteration of short-chain fatty acids (SCFAs) content and the damage to the intestinal barrier. However, the mediating effect of IM on intestine dysfunction is largely unknown. Herein, we conducted comprehensive intestinal microbiota transplantation (IMT) to explore the link between IM and SUS development. Furthermore, we isolated and identified a strain with an ability to produce acetic acid from both healthy individual and SUS individual with IM from healthy donors. We found that dysbiotic IM and intestinal barrier function in SUS recipients could be restored by IM from healthy donors. The strain could restore IM community and intestinal barrier function. Consistently, acetate supply also restores intestinal homeostasis of SUS-diseased and -infected . Mechanically, acetate was found to specifically bind to its receptor-free fatty acid receptor 2 (FFAR2) to mediate IM structure community and intestinal barrier function. Knockdown of FFAR2 by transfection of specific FFAR2 siRNA could hamper acetate-mediated intestinal homeostasis . Furthermore, we confirmed that acetate/FFAR2 could inhibit -activated NF-κB-MLCK-MLC signaling pathway to restore intestinal epithelium integrity and upregulated the expression of ZO-1 and Occludin. Our findings provide the first evidence that restores pathogen-induced intestinal barrier dysfunction via acetate/FFAR2-NF-κB-MLCK-MLC axis, which provides new insights into the control and prevention of SUS outbreak from an ecological perspective.IMPORTANCESkin ulceration syndrome (SUS) as a main disease in aquaculture has severely restricted the developmental aquaculture industry. Intestinal microbiota (IM) has been studied extensively due to its immunomodulatory properties. Short-chain fatty acids (SCFAs) as an essential signal molecule for microbial regulation of host health also have attracted wide attention. Therefore, it is beneficial to explore the link between IM and SUS for prevention and control of SUS. In the study, the contribution of IM to SUS development has been examined. Additionally, our research further validated the restoration of SCFAs on intestinal barrier dysfunction caused by SUS via isolating SCFAs-producing bacteria. Notably, this restoration might be achieved by inhibition of NF-κB-MLCK-MLC signal pathway, which could be activated by . These findings may have important implications for exploration of the role of IM in SUS occurrence and provide insight into the SUS treatment.
PubMed: 38940521
DOI: 10.1128/msystems.00602-24