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In recent decades, there has been an increase in the occurrence of fungal infections; yet, the arsenal of drugs available to fight invasive infections remains very limited. The development of new antifungal agents is hindered by the restricted number of molecular targets that can be exploited, given the shared eukaryotic nature of fungi and their hosts which often leads to host toxicity. In this paper, we examine the riboflavin biosynthetic pathway as a potential novel drug target. Riboflavin is an essential nutrient for all living organisms. Its biosynthetic pathway does not exist in humans, who obtain riboflavin through their diet. Our findings demonstrate that all enzymes in the pathway are essential for Candida albicans, Candida glabrata, and Saccharomyces cerevisiae. Auxotrophic strains, which mimic a drug targeting the biosynthesis pathway, experience rapid mortality in the absence of supplemented riboflavin. Furthermore, RIB1 is essential for virulence in both C. albicans and C. glabrata in a systemic mouse model. The fungal burden of a RIB1 deletion strain is significantly reduced in the kidneys and brain of infected mice, and this reduction becomes more pronounced over time. Nevertheless, auxotrophic cells can still take up external riboflavin when supplemented. We identified Orf19.4337 as the riboflavin importer in C. albicans and named it Rut1. We found that Rut1 only facilitates growth at external riboflavin concentrations that exceed the physiological concentrations in the human body. This suggests that riboflavin uptake is unlikely to serve as a resistance mechanism against drugs targeting the biosynthesis pathway. Interestingly, the uptake system in S. cerevisiae is more effective than in C. albicans and C. glabrata, enabling an auxotrophic S. cerevisiae strain to outcompete an auxotrophic C. albicans strain in lower riboflavin concentrations.

Importance: Candida species are a common cause of invasive fungal infections. Candida albicans, in particular, poses a significant threat to immunocompromised individuals. This opportunistic pathogen typically lives as a commensal on mucosal surfaces of healthy individuals but it can also cause invasive infections associated with high morbidity and mortality. Currently, there are only three major classes of antifungal drugs available to treat these infections. In addition, the efficacy of these antifungal agents is restricted by host toxicity, suboptimal pharmacokinetics, a narrow spectrum of activity, intrinsic resistance of fungal species, such as Candida glabrata, to certain drugs, and the acquisition of resistance over time. Therefore, it is crucial to identify new antifungal drug targets with novel modes of action to add to the limited armamentarium.

Hepatitis E virus (HEV) is distinct from other hepatotropic viruses because it is zoonotic. HEV-1 and HEV-2 exclusively infect humans, whereas HEV-3 and HEV-4 are zoonotic. However, the viral and/or host factors responsible for cross-species HEV transmission remain elusive. The hypervariable region (HVR) in HEV is extremely heterogenetic and is implicated in HEV adaptation. Here, we investigated the potential role of Serine phosphorylation in the HVR in HEV replication. We first analyzed HVR sequences across different HEV genotypes and identified a unique region at the N-terminus of the HVR, which is variable in the human-exclusive HEV genotypes but relatively conserved in zoonotic HEV genotypes. Using predictive tools, we identified four potential phosphorylation sites that are highly conserved in zoonotic HEV-3 and HEV-4 genomes but absent in human-exclusive HEV-1 strains. To explore the functional significance of these putative phosphorylation sites, we introduced mutations into the HEV-3 infectious clone and indicator replicon, replacing each Serine residue individually with alanine or aspartic acid, and assessed the impact of these substitutions on HEV-3 replication. We found that the phospho-blatant S711A mutant significantly reduced virus replication, whereas the phospho-mimetic S711D mutant modestly reduced virus replication. Conversely, mutations in the other three Serine residues did not significantly affect HEV-3 replication. Furthermore, we demonstrated that Ser711 phosphorylation did not alter host cell tropism of zoonotic HEV-3. In conclusion, our results showed that potential phosphorylation of the Ser711 residue significantly affects HEV-3 replication in vitro, providing new insights into the potential mechanisms of zoonotic HEV transmission.IMPORTANCEHEV is an important zoonotic pathogen, causing both acute and chronic hepatitis E and extrahepatic manifestation of diseases, such as neurological sequelae. The zoonotic HEV-3 is linked to chronic infection and neurological diseases. The specific viral and/or host factors facilitating cross-species HEV infection are unknown. The intrinsically disordered HVR in ORF1 is crucial for viral fitness and adaptation, both in vitro and in vivo. We hypothesized that phosphorylation of Serine residues in the HVR of zoonotic HEV by unknown host cellular kinases is associated with cross-species HEV transmission. In this study, we identified a conserved region within the HVR of zoonotic HEV strains but absent in the human-exclusive HEV-1 and HEV-2. We elucidated the important role of phosphorylation at the Ser711 residue in zoonotic HEV-3 replication, without altering the host cell tropism. These findings contribute to our understanding the mechanisms of cross-species HEV transmission.

Though a confined or a broad population is exposed respectively to endemic or pandemic infections, in the same environment, some individuals resist the development of infections. The attributed reason is the inheritance of a set of immune system genes that can efficiently deal with the pathogens. In this study, we show how outbred mice differentially respond to Cryptococcus neoformans, a fungal pathogen, and the mechanism through which the surviving mice mount a protective immune defense. We identified that those mice developing antibodies specifically against Pep1p, an aspartic protease secreted by C. neoformans, had significantly improved survival. Vaccination (either prophylactic or therapeutic) with a recombinant Pep1p significantly increased the survival of the mice by decreasing the fungal load and stimulating a protective immune response. Passive immunization of C. neoformans-infected mice with monoclonal antibodies developed against Pep1p also improves the survival of the mice by increasing phagocytosis of C. neoformans and decreasing the multiplication of this fungus. Together, these data demonstrate the prophylactic and therapeutic potentials of the C. neoformans antigenic protein Pep1p or Pep1p-specific antibodies against this fungal infection. Also, this study suggests that the immunological interaction and thereby the responses developed against a pathogen guide the hosts to behave differentially against microbial pathogenicity.

Importance: Vaccination and immunotherapies against fungal pathogens still remain a challenge. Here, we show using an in vivo model based on outbred mice that development of antibodies against Pep1p, an antigenic protein of the fungal pathogen Cryptococcus neoformans, confers resistance to this fungal infection. In support of this observation, prophylactic or therapeutic immunization of the mice with recombinant Pep1p could improve their survival when infected with a lethal dose of C. neoformans. Moreover, passive therapy with monoclonal anti-Pep1p antibodies also enhanced survival of the mice from C. neoformans infection. The associated antifungal mechanisms were mounting of a protective immune response and the development of fungal specific antibodies that decrease the fungal burden due to an increase in their phagocytosis and/or inhibit the fungal multiplication. Together, our study demonstrates (a) the mode of host-fungal interaction and the immune response developed thereby play a crucial role in developing resistance against C. neoformans; (b) Pep1p, an aspartic protease as well as an antigenic protein secreted by C. neoformans, can be exploited for vaccination (both prophylactic and therapeutic) or immunotherapy to improve the host defense during this fungal infection.

The gut microbiota plays a critical role in human health and disease. Microbial community assembly and succession early in life are influenced by numerous factors. In turn, assembly of this microbial community is known to influence the host, including immune system development, and has been linked to outcomes later in life. To date, the role of host-mediated nutritional immunity and metal availability in shaping microbial community assembly and succession early in life has not been explored in depth. Using a human infant cohort, we show that the metal-chelating protein calprotectin is highly abundant in infants. Taxa previously shown to be successful early colonizers of the infant gut, such as Enterococcus, Enterobacteriaceae, and Bacteroides, are highly resistant to experimental metal starvation in culture. Lactobacillus, meanwhile, is highly susceptible to metal restriction, pointing to a possible mechanism by which host-mediated metal limitation shapes the fitness of early colonizing taxa in the infant gut. We further demonstrate that formula-fed infants harbor markedly higher levels of metals in their gastrointestinal tract compared to breastfed infants. Formula-fed infants with high levels of metals harbor distinct microbial communities compared to breastfed infants, with higher levels of Enterococcus, Enterobacter, and Klebsiella, taxa which show increased resistance to the toxic effects of high metal concentrations. These data highlight a new paradigm in microbial community assembly and suggest an unappreciated role for nutritional immunity and dietary metals in shaping the earliest colonization events of the microbiota.IMPORTANCEEarly life represents a critical window for microbial colonization of the human gastrointestinal tract. Surprisingly, we still know little about the rules that govern the successful colonization of infants and the factors that shape the success of early life microbial colonizers. In this study, we report that metal availability is an important factor in the assembly and succession of the early life microbiota. We show that the host-derived metal-chelating protein, calprotectin, is highly abundant in infants and successful early life colonizers can overcome metal restriction. We further demonstrate that feeding modality (breastmilk vs formula) markedly impacts metal levels in the gut, potentially influencing microbial community succession. Our work suggests that metals, a previously unexplored aspect of early life ecology, may play a critical role in shaping the early events of microbiota assembly in infants.

Toxoplasma gondii possesses a highly polarized secretory pathway that contains both broadly conserved eukaryotic organelles and unique apicomplexan organelles, which play essential roles in the parasite's lytic cycle. As in other eukaryotes, the T. gondii Golgi apparatus sorts and modifies proteins prior to their distribution to downstream organelles. Many of the typical trafficking factors found involved in these processes are missing from apicomplexan genomes, suggesting that these parasites have evolved unique proteins to fill these roles. Here, we identify a Golgi-localizing protein (ULP1), which is structurally similar to the eukaryotic trafficking factor p115/Uso1. We demonstrate that depletion of ULP1 leads to a dramatic reduction in parasite fitness that is the result of defects in microneme secretion, invasion, replication, and egress. Using ULP1 as bait for TurboID proximity labeling and immunoprecipitation, we identify 11 more Golgi-associated proteins and demonstrate that ULP1 interacts with the T. gondii-conserved oligomeric Golgi (COG) complex. These proteins include both conserved trafficking factors and parasite-specific proteins. Using a conditional knockdown approach, we assess the effect of each of these 11 proteins on parasite fitness. Together, this work reveals a diverse set of T. gondii Golgi-associated proteins that play distinct roles in the secretory pathway. As several of these proteins are absent outside of the Apicomplexa, they represent potential targets for the development of novel therapeutics against these parasites.

Importance: Apicomplexan parasites such as Toxoplasma gondii infect a large percentage of the world's population and cause substantial human disease. These widespread pathogens use specialized secretory organelles to infect their host cells, modulate host cell functions, and cause disease. While the functions of the secretory organelles are now better understood, the Golgi apparatus of the parasite remains largely unexplored, particularly regarding parasite-specific innovations that may help direct traffic intracellularly. In this work, we characterize ULP1, a protein that is unique to parasites but shares structural similarity to the eukaryotic trafficking factor p115/Uso1. We show that ULP1 plays an important role in parasite fitness and demonstrate that it interacts with the conserved oligomeric Golgi (COG) complex. We then use ULP1 proximity labeling to identify 11 additional Golgi-associated proteins, which we functionally analyze via conditional knockdown. This work expands our knowledge of the Toxoplasma Golgi apparatus and identifies potential targets for therapeutic intervention.

Candida auris, a multidrug-resistant human fungal pathogen, was first identified in 2009 in Japan. Since then, systemic C. auris infections have now been reported in more than 50 countries, with mortality rates of 30%-60%. A major contributing factor to its high inter- and intrahospital clonal transmission is that C. auris, unlike most Candida species, displays unique skin tropism and can stay on human skin for a prolonged period. However, the molecular mechanisms responsible for C. auris skin colonization, intradermal persistence, and systemic virulence are poorly understood. Here, we report that C. auris Hog1 mitogen-activated protein kinase is essential for efficient skin colonization, intradermal persistence as well as systemic virulence. RNA-seq analysis of wild-type parental and hog1Δ mutant strains revealed marked downregulation of genes involved in processes such as cell adhesion, cell wall rearrangement, and pathogenesis in hog1Δ mutant compared to the wild-type parent. Consistent with these data, we found a prominent role for Hog1 in maintaining cell wall architecture, as the hog1Δ mutant demonstrated a significant increase in cell-surface β-glucan exposure and a concomitant reduction in chitin content. Additionally, we observed that Hog1 was required for biofilm formation in vitro and fungal survival when challenged with primary murine macrophages and neutrophils ex vivo. Collectively, these findings have important implications for understanding the C. auris skin adherence mechanisms and penetration of skin epithelial layers preceding bloodstream infections.

Importance: Candida auris is a World Health Organization fungal priority pathogen and an urgent public health threat recognized by the Centers for Disease Control and Prevention. C. auris has a unique ability to colonize human skin. It also persists on abiotic surfaces in healthcare environments for an extended period of time. These attributes facilitate the inter- and intrahospital clonal transmission of C. auris. Therefore, understanding C. auris skin colonization mechanisms is critical for infection control, especially in hospitals and nursing homes. However, despite its profound clinical relevance, the molecular and genetic basis of C. auris skin colonization mechanisms are poorly understood. Herein, we present data on the identification of the Hog1 MAP kinase as a key regulator of C. auris skin colonization. These findings lay the foundation for further characterization of unique mechanisms that promote fungal persistence on human skin.

In bacteria, if a ribosome translates an mRNA lacking a stop codon it becomes stalled at the 3' end of the message. These ribosomes must be rescued by trans-translation or the alternative rescue factors (ArfA or ArfB). However, mounting evidence suggests that the ribosome quality control (RQC) pathway may also rescue non-stop ribosomes. Here, we surveyed the conservation of ribosome rescue pathways in >15,000 bacterial genomes. We found that trans-translation is conserved in >97% of bacterial genomes, while the other rescue pathways are restricted to particular phyla. We did not detect the gene encoding RqcH, the major mediator of RQC, in Proteobacteria (Pseudomonadota). In all Proteobacteria investigated to date, trans-translation is essential in the absence of the Arf proteins. Therefore, we tested whether expression of RQC components from Bacillus subtilis could rescue viability in the absence of trans-translation and ArfA in Escherichia coli. We found that the RQC pathway indeed functions in E. coli and rescues the well-documented synthetic lethal phenotype of ∆ssrA∆arfA. Moreover, we show that the RQC pathway in B. subtilis is essential in the absence of trans-translation and ArfA, further supporting a role for the RQC pathway in the rescue of non-stop ribosomes. Finally, we report a strong co-occurrence between RqcH and the ribosome splitting factor MutS2, but present experimental evidence that there are likely additional ribosome splitting factors beyond MutS2 in B. subtilis. Altogether, our work supports a role for RQC in non-stop ribosome rescue and provides a broad survey of ribosome rescue pathways in diverse bacteria.

Importance: In bacteria, it is estimated that 2%-4% of all translation reactions terminate with the ribosome stalled on a damaged mRNA lacking a stop codon. Mechanisms that rescue these ribosomes are essential for viability. We determined the functional overlap between the ribosome quality control pathway and the classical non-stop rescue systems [alternative rescue factor (ArfA) and trans-translation] in a representative Firmicute and Proteobacterium, phyla that are evolutionarily distinct. Furthermore, we used a bioinformatics approach to examine the conservation and overlap of various ribosome rescue systems in >15,000 species throughout the bacterial domain. These results provide key insights into ribosome rescue in diverse phyla.

After centuries of relative stability, the scientific publishing world has undergone tremendous disruption and change during the first decades of the 21st century. The causes for disruption can be traced to the information revolution, which brought such benefits as rapid publication, greater connectivity, and ready access to large databases, along with less desirable practices including image manipulation, plagiarism, and other ethical transgressions. The information revolution has driven the proliferation of journals, expansion of for-profit academic publishing, and empowerment of the open-access movement, each of which has exerted new financial pressures on traditional publishing models. As journals became the focal point for ethical concerns in science, they have adapted by increasing the scope of their duties, which now include archiving of data, enforcement of good practices, establishment of standards for rigor, and training the next generation of reviewers and editors. Here, we consider the seismic changes occurring in scientific publishing and place them into the context of a rapidly changing landscape of scientific and publishing norms.