Multiple Pathogenic Species Research Articles

Limit of detection of Salmonella ser. Enteritidis using culture-based versus culture-independent diagnostic approaches.

The appeal of culture-independent diagnostic tests (CIDTs) is increased speed with lowered cost, as well as the potential to detect multiple pathogen species in a single analysis and to monitor other areas of concern such as antimicrobial resistance genes or virulence factors. This study provides quantitative data on the sensitivity of CIDTs relative to current approaches, which is essential for determining the feasibility of implementing these methods in pathogen surveillance programs.

Genus-targeted markers for the taxonomic identification and monitoring of coagulase-positive and coagulase-negative Staphylococcus species

The Staphylococcus genus comprises multiple pathogenic and opportunistic species that represent a risk to public health. Epidemiological studies require accurate taxonomic classification of isolates with enough resolution to distinguish clonal complexes. Unfortunately, 16 S rRNA molecular analysis and phenotypic characterization cannot distinguish all species and do not offer enough resolution to assess intraspecific diversity. Other approaches, such as Multilocus Sequence Tagging, provide higher resolution; however, they have been developed for Staphylococcus aureus and a few other species. Here, we developed a set of genus-targeted primers using five orthologous genes (pta, tuf, tpi, groEs, and sarA) to identify all Staphylococcus species within the genus. The primers were initially evaluated using 20 strains from the Collection of Microorganisms of Interest in Animal Health from AGROSAVIA (CMISA), and their amplified sequences were compared to a set of 33 Staphylococcus species. This allowed the taxonomic identification of the strains even on close species and the establishment of intraspecies diversity. To enhance the scope and cost-effectiveness of the proposed strategy, we customized the primer sets for an Illumina paired-end amplicon protocol, enabling gene multiplexing. We assessed five genes across 177 strains, generating 880 paired-end libraries from the CMISA. This approach significantly reduced sequencing costs, as all libraries can be efficiently sequenced in a single MiSeq run at a fraction (one-fourth or less) of the cost associated with Sanger sequencing. In summary, this method can be used for precise identification and diversity analysis of Staphylococcus species, offering an advancement over traditional techniques in both resolution and cost-effectiveness.Graphical

Functional surface expression of immunoglobulin cleavage systems in a candidate Mycoplasma vaccine chassis

The Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system is a candidate ‘virulence factor present in multiple pathogenic species of the Mollicutes, including the fast-growing species Mycoplasma feriruminatoris. The MIB-MIP system cleaves the heavy chain of host immunoglobulins, hence affecting antigen-antibody interactions and potentially facilitating immune evasion. In this work, using -omics technologies and 5’RACE, we show that the four copies of the M. feriruminatoris MIB-MIP system have different expression levels and are transcribed as operons controlled by four different promoters. Individual MIB-MIP gene pairs of M. feriruminatoris and other Mollicutes were introduced in an engineered M. feriruminatoris strain devoid of MIB-MIP genes and were tested for their functionality using newly developed oriC-based plasmids. The two proteins are functionally expressed at the surface of M. feriruminatoris, which confirms the possibility to display large membrane-associated proteins in this bacterium. However, functional expression of heterologous MIB-MIP systems introduced in this engineered strain from phylogenetically distant porcine Mollicutes like Mesomycoplasma hyorhinis or Mesomycoplasma hyopneumoniae could not be achieved. Finally, since M. feriruminatoris is a candidate for biomedical applications such as drug delivery, we confirmed its safety in vivo in domestic goats, which are the closest livestock relatives to its native host the Alpine ibex.

Phytophthora and Phytopythium species associated with walnut dieback in plantations of northern Patagonia, Argentina

AbstractThe walnut industry in Argentina has grown in recent years, especially in northern Patagonia. However, this expansion has also brought new challenges, including the emergence of diseases threatening the health and productivity of walnut plantations. One of the most important diseases affecting walnut crops worldwide is ‘ink disease’ or ‘walnut dieback'. The aim of this study was to characterize ink disease, identify the aetiological agents, and estimate its incidence in walnut orchards of northern Patagonia, Argentina. Symptomatic trees were observed in the seven surveyed walnut orchards, showing a range of symptoms, including chlorosis and wilting, stem cankers and desiccation. In vivo assay and molecular analyses revealed that not only Phytophthora (P.) but also Phytopythium (Ph.) species are the causal agents of the disease, with P. drechsleri, Ph. mercuriale and Ph. vexans being the most virulent over the commonly used rootstock Juglans hindsii. Notably, Ph. vexans was the most frequently isolated pathogen, suggesting its prevalence and potential impact on the regional walnut industry. Overall, this study provides critical insights into the prevalence and impact of Phytophthora and Phytopythium species on walnut health in northern Patagonia. The presence of multiple pathogenic oomycete species raises concerns about the health of walnut orchards, as well as the potential risk of pathogen transmission to other fruit crops in the region. The findings will help growers to develop better cultural practices and implement measures to guarantee the sustainability of the walnut industry in Patagonia.

Identification and Characterization of Pythium deliense Causing Sugar Beet (Beta vulgaris) Root Rot in Grand Forks, North Dakota

Damping-off disease caused by multiple species of soilborne pathogens causes severe economic losses to sugar beet production. Thus, identifying the causal agent is important to formulating effective disease management. During a July 2021 disease survey in a sugar beet field at Grand Forks, North Dakota, we observed that approximately 15% of plants located in low areas of a commercial field showed wilting leaves that were pale green and slightly blighted in color, and the roots were dark brown to black, along with irregular necrotic lesions. The isolated pathogen had the characteristic morphology of Pythium deliense. The sequences of the internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit 1 mitochondrial ( cox1) genes were 100% homologous to corresponding sequences in the NCBI database, which confirmed its P. deliense identity. Moreover, the ITS phylogeny confirmed its identity as P. deliense and its close relationship with P. aphanidermatum. A disease screening trial with four sugar beet varieties was done. Growers and breeders could benefit from accurate information about the sugar beet pathogen in a given field to formulate effective disease management practices.

Deciphering salivary microbiome signature in Crohn’s disease patients with different factors contributing to dysbiosis

Crohn's disease (CD) is a chronic inflammatory bowel disease. An imbalanced microbiome (dysbiosis) can predispose to many diseases including CD. The role of oral dysbiosis in CD is poorly understood. We aimed to explore microbiome signature and dysbiosis of the salivary microbiome in CD patients, and correlate microbiota changes to the level of inflammation. Saliva samples were collected from healthy controls (HC) and CD patients (n = 40 per group). Salivary microbiome was analyzed by sequencing the entire 16S rRNA gene. Inflammatory biomarkers (C-reactive protein and calprotectin) were measured and correlated with microbiome diversity. Five dominant species were significantly enriched in CD, namely Veillonella dispar, Megasphaera stantonii, Prevotella jejuni, Dolosigranulum pigrum and Lactobacillus backii. Oral health had a significant impact on the microbiome since various significant features were cariogenic as Streptococcus mutans or periopathogenic such as Fusobacterium periodonticum. Furthermore, disease activity, duration and frequency of relapses impacted the oral microbiota. Treatment with monoclonal antibodies led to the emergence of a unique species called Simonsiella muelleri. Combining immunomodulatory agents with monoclonal antibodies significantly increased multiple pathogenic species such as Salmonella enterica, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Loss of diversity in CD was shown by multiple diversity indices. There was a significant negative correlation between gut inflammatory biomarkers (particularly calprotectin) and α-diversity, suggesting more inflammation associated with diversity loss in CD. Salivary dysbiosis was evident in CD patients, with unique microbiota signatures and perturbed species that can serve as disease biomarkers or potential targets for microbiota modulation. The interplay of various factors collectively contributed to dysbiosis, although each factor probably had a unique effect on the microbiome. The emergence of pathogenic bacteria in the oral cavity of CD patients is alarming since they can disturb gut homeostasis and induce inflammation by swallowing, or hematogenous spread of microbiota, their metabolites, or generated inflammatory mediators.

Reconstructing multi-strain pathogen interactions from cross-sectional survey data via statistical network inference.

Infectious diseases often involve multiple pathogen species or multiple strains of the same pathogen. As such, knowledge of how different pathogens interact is key to understand and predict the outcome of interventions targeting only a subset of species or strains involved in disease. Population-level data may be useful to infer pathogen strain interactions, but most previously used inference methods only consider uniform interactions between all strains or focus on marginal pairwise interactions. As such, these methods are prone to bias induced by indirect interactions through other strains. Here, we evaluated statistical network inference for reconstructing heterogeneous interactions from cross-sectional surveys detecting joint presence/absence patterns of pathogen strains within hosts. We applied various network models to simulated survey data, representing endemic infection states of multiple pathogen strains with potential interactions in acquisition or clearance of infection. Satisfactory performance was demonstrated by the estimators converging to the true interactions. Accurate reconstruction of interaction networks was achieved by regularization or penalization for sample size. Although performance deteriorated in the presence of host heterogeneity, this was overcome by correcting for individual-level risk factors. Our work demonstrates how statistical network inference could prove useful for detecting multi-strain pathogen interactions and may have applications beyond epidemiology.

Investigating Possible Interspecies Communication of Plasmids Associated with Transfer of Third-Generation Cephalosporin, Quinolone, and Colistin Resistance Between Simultaneously Isolated Escherichia Coli and Klebsiella Pneumoniae.

The coinfection process producing multiple species of pathogens provides a specific ecological niche for the exchange of genetic materials between pathogens, in which plasmids play a vital role in horizontal gene transfer, especially for drug resistance, but the underlying transfer pathway remains unclear. Interspecies communication of the plasmids associated with the transfer of third-generation cephalosporins, quinolones, and colistin resistance has been observed in simultaneously isolated Escherichia coli and Klebsiella pneumoniae from abdominal drainage following surgery. The MICs of antimicrobial agents were determined by the broth microdilution method. The complete chromosome and plasmid sequences were obtained by combining Illumina paired-end short reads and MinION long reads. S1-PFGE, southern blot analysis and conjugation assay confirmed the transferability of the mcr-1-harboring plasmid. Both the E. coli isolate EC15255 and K. pneumoniae isolate KP15255 from the same specimen presented multidrug resistance. Each of them harbored one chromosome and three plasmids, and two plasmids and their mediated resistance could be transferred to the recipient by conjugation. Comparison of their genome sequences suggested that several genetic communication events occurred between species, especially among their plasmids, such as whole-plasmid transfer, insertion, deletion, amplification, or inversion. Exchange of plasmids or the genetic elements they harbor plays a critical role in antimicrobial resistance gene transmission and poses a substantial threat to nosocomial infection control, necessitating the continued surveillance of multidrug resistant pathogens, especially during coinfection. IMPORTANCE The genome sequence of bacterial pathogens commonly provides a detailed clue of genetic communication among clones or even distinct species. The intestinal microecological environment is a representative ecological niche for genetic communication. However, it is still difficult to describe the details of horizontal gene transfer or other genetic events within them because the evidence in the genome sequence is incomplete and limited. In this study, the simultaneously isolated Escherichia coli and Klebsiella pneumoniae from a coinfection process provided an excellent example for observation of interspecies communication between the two genomes and the plasmids they harbor. A complete genome sequence acquired by combining the Illumina and MinION sequencing platforms facilitated the understanding of genetic communication events, such as whole-plasmid transfer, insertion, deletion, amplification, or inversion, which contribute to antimicrobial resistance gene transmission and are a substantial threat to nosocomial infection control.

Specific resistance prevents the evolution of general resistance and facilitates disease emergence.

Host-shifts, where pathogens jump from an ancestral host to a novel host, can be facilitated or impeded by standing variation in disease resistance, but only if resistance provides broad-spectrum general resistance against multiple pathogen species. Host resistance comes in many forms and includes both general resistance, as well as specific resistance, which may only be effective against a single pathogen species or even genotype. However, most evolutionary models consider only one of these forms of resistance, and we have less understanding of how these two forms of resistance evolve in tandem. Here, we develop a model that allows for the joint evolution of specific and general resistance and asks if the evolution of specific resistance drives a decrease in the evolution of general resistance. We also explore how these evolutionary outcomes affect the risk of foreign pathogen invasion and persistence. We show that in the presence of a single endemic pathogen, the two forms of resistance are strongly exclusionary. Critically, we find that specific resistance polymorphisms can prevent the evolution of general resistance, facilitating the invasion of foreign pathogens. We also show that specific resistance polymorphisms are a necessary condition for the successful establishment of foreign pathogens following invasion, as they prevent the exclusion of the foreign pathogen by the more transmissible endemic pathogen. Our results demonstrate the importance of considering the joint evolution of multiple forms of resistance when evaluating a population's susceptibility to foreign pathogens.

Combining Acoustic Bioprinting with AI-Assisted Raman Spectroscopy for High-Throughput Identification of Bacteria in Blood.

Identifying pathogens in complex samples such as blood, urine, and wastewater is critical to detect infection and inform optimal treatment. Surface-enhanced Raman spectroscopy (SERS) and machine learning (ML) can distinguish among multiple pathogen species, but processing complex fluid samples to sensitively and specifically detect pathogens remains an outstanding challenge. Here, we develop an acoustic bioprinter to digitize samples into millions of droplets, each containing just a few cells, which are identified with SERS and ML. We demonstrate rapid printing of 2 pL droplets from solutions containing S. epidermidis, E. coli, and blood; when they are mixed with gold nanorods (GNRs), SERS enhancements of up to 1500× are achieved.We then train a ML model and achieve ≥99% classification accuracy from cellularly pure samples and ≥87% accuracy from cellularly mixed samples. We also obtain ≥90% accuracy from droplets with pathogen:blood cell ratios <1. Our combined bioprinting and SERS platform could accelerate rapid, sensitive pathogen detection in clinical, environmental, and industrial settings.

Infectious disease ecology and evolution in a changing world.

Infectious disease ecology and evolution in a changing world.

A natural allele of proteasome maturation factor improves rice resistance to multiple pathogens.

Crops with broad-spectrum resistance loci are highly desirable in agricultural production because these loci often confer resistance to most races of a pathogen or multiple pathogen species. Here we discover a natural allele of proteasome maturation factor in rice, UMP1R2115, that confers broad-spectrum resistance to Magnaporthe oryzae, Rhizoctonia solani, Ustilaginoidea virens and Xanthomonas oryzae pv. oryzae. Mechanistically, this allele increases proteasome abundance and activity to promote the degradation of reactive oxygen species-scavenging enzymes including peroxidase and catalase upon pathogen infection, leading to elevation of H2O2 accumulation for defence. In contrast, inhibition of proteasome function or overexpression of peroxidase/catalase-encoding genes compromises UMP1R2115-mediated resistance. More importantly, introduction of UMP1R2115 into a disease-susceptible rice variety does not penalize grain yield while promoting disease resistance. Our work thus uncovers a broad-spectrum resistance pathway integrating de-repression of plant immunity and provides a valuable genetic resource for breeding high-yield rice with multi-disease resistance.

Protection of rainbow trout (Oncorhynchus mykiss) against VHSV genotype Ia and IHNV by immunization with VHSV genotype IVa backbone-based single-cycle viruses

To evaluate the protective effect of viral hemorrhagic septicemia virus genotype IVa (VHSV IVa) genome-based single-cycle viruses against VHSV genotype Ia (VHSV Ia) and infectious hematopoietic necrosis virus (IHNV) in rainbow trout, three kinds of single-cycle VHSVs were rescued using reverse genetic technology: i) rVHSV-IaGΔTM-IVaG containing the transmembrane and cytoplasmic region-deleted G protein (GΔTM) of VHSV Ia instead of VHSV IVa full G gene ORF and having VHSV IVa G proteins on the envelope; ii) rVHSV-IaGΔTM-IaG containing VHSV Ia GΔTM instead of VHSV IVa full G gene ORF and having VHSV Ia G proteins on the envelope; iii) rVHSV-IaGΔTM-ihnvGΔTM-IVaG containing not only VHSV Ia GΔTM instead of full G gene but also IHNV GΔTM instead of NV gene and having VHSV IVa G proteins on the envelope. Rainbow trout immunized with rVHSV-IaGΔTM-IaG and rVHSV-IaGΔTM-IVaG showed significantly higher serum antibody titers against both VHSV Ia and VHSV IVa, and showed no mortality against VHSV Ia infection, while fish in the control groups showed 100% mortalities. Fish immunized with rVHSV-IaGΔTM-ihnvGΔTM-IVaG showed significantly higher serum antibody titers against VHSV IVa, VHSV Ia, and IHNV compared to fish in the control group. Immunization with rVHSV-IaGΔTM-ihnvGΔTM-IVaG induced significantly higher protection against not only VHSV Ia but also IHNV. These results suggest that the present single-cycle rVHSV-based system can be used as a platform to produce combined vaccines that can protect fish from multiple pathogenic species. However, the mechanism of the high protection against IHNV despite comparatively low antibody titer remains to be investigated.

Development and application of multiplex targeted-sequencing approaches to identify Phytophthora species associated with root rot and wilting complex of red raspberry.

Phytophthora species are primary causal agents of raspberry root rot and wilting complex (RRWC), a disease complex that is of major concern to raspberry producers worldwide. Accurate identification of the causal agents is a first step for effective disease management. Advancements in molecular diagnostics can facilitate the detection of multiple pathogen species associated with this disease complex. We developed multiplex targeted-sequencing methods using degenerate primers for heat shock protein 90, elongation factor 1α and β-tubulin genes to identify Phytophthora species causing RRWC. One hundred and twenty-eight isolates recovered during 2018 to 2020 from diverse fields in major raspberry growing areas of British Columbia (BC) were sequenced and identified by comparing with known reference sequences of 142 Phytophthora species, 111 Pythium species, and nine Phytopythium species in the NCBI database. This multiplex targeted-sequencing method was highly specific and identified two species of Phytophthora associated with RRWC. These were P. rubi (85% of isolates) and P. gonapodyides (15% of isolates). Phytophthora rubi was predominantly isolated from the cultivars 'Chemainus' (51%), 'Rudi' (27%) and 'Meeker' (15%), whereas P. gonapodyides was predominately isolated from the moderately resistant cultivar 'Cascade Bounty'. Pathogenicity studies on intact plants and detached leaves confirmed that P. rubi and P. gonapodyides can cause symptoms of RRWC on raspberry, thus fulfilling Koch's postulates. To our knowledge, this is the first report of P. gonapodyides as a causal agent of RRWC on raspberry in BC. This study provides novel insights into the identification and species composition of Phytophthora associated with RRWC in raspberry production systems.

Dual phage amplification-mediated multiplex detection strategies for the simultaneous detection of Salmonella enterica and Staphylococcus aureus

Multiplex bacteria detection is essential to monitor various pathogens situations where multiple pathogen species are a concern. Herein, we explored novel dual phage amplification-based strategies (PAA) combined with a multiplex detection technique for simultaneous bacteria detection in one single tube. This assay was normalized on two common foodborne pathogens: Salmonella enterica and Staphylococcus aureus. In the presence of the target bacteria, phages could specifically recognize the viable cells and replicate themselves within the cells to produce more progeny phages. After thermal lysis within 10 min, the products with DNA of the progeny phages were analyzed with multiplex detection strategies. The specificity of the multiplex detection strategies integrated with the high sensitivity of the PAA assay allowed this assay to be rapid, specific, and sensitive for simultaneous quantification of Salmonella and Staphylococcus aureus. Optimized parameters of PAA, single and multiplex qPCR were defined, and the analytical sensitivities, specificities, and stabilities of single and multiplex reactions were validated under defined experimental conditions and in real samples. The results demonstrated wide linear ranges (10–108 CFU/mL), with detection limits of 10 CFU/mL for all bacteria tested. Moreover, the total detection time could be reduced to no more than 4 h. This assay was successfully applied to real food samples consistent with the results comparable to traditional plate counting methods. Due to the rapid and simple operation, low sensitivity and cost, high specificity, and the ability to distinguish live bacteria, the proposed PAA-based multiplex qPCR assay provides an effective and promising strategy for the simultaneous detection of viable bacteria.

Multiplexed LNP-mRNA vaccination against pathogenic coronavirus species.

Although COVID-19 vaccines have been developed, multiple pathogenic coronavirus species exist, urging on development of multispecies coronavirus vaccines. Here we develop prototype lipid nanoparticle (LNP)-mRNA vaccine candidates against SARS-CoV-2 Delta, SARS-CoV, and MERS-CoV, and we test how multiplexing LNP-mRNAs can induce effective immune responses in animal models. Triplex and duplex LNP-mRNA vaccinations induce antigen-specific antibody responses against SARS-CoV-2, SARS-CoV, and MERS-CoV. Single-cell RNA sequencing profiles the global systemic immune repertoires and respective transcriptome signatures of vaccinated animals, revealing a systemic increase in activated B cells and differential gene expression across major adaptive immune cells. Sequential vaccination shows potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrate the feasibility, antibody responses, and single-cell immune profiles of multispecies coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights into optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species.

An evolutionarily conserved C4HC3-type E3 ligase regulates plant broad-spectrum resistance against pathogens.

Deployment of broad-spectrum disease resistance against multiple pathogen species is an efficient way to control plant diseases. Here, we identify a Microtubule-associated C4HC3-type E3 Ligase (MEL) in both Nicotiana benthamiana and Oryza sativa, and show that it is able to integrate and initiate a series of host immune signaling, conferring broad-spectrum resistance to viral, fungal, and bacterial pathogens. We demonstrate that MEL forms homodimer through intermolecular disulfide bonds between its cysteine residues in the SWIM domain, and interacts with its substrate serine hydroxymethyltrasferase 1 (SHMT1) through the YφNL motif. Ubiquitin ligase activity, homodimerization and YφNL motif are indispensable for MEL to regulate plant immunity by mediating SHMT1 degradation through the 26S proteasome pathway. Our findings provide a fundamental basis for utilizing the MEL-SHMT1 module to generate broad-spectrum-resistant rice to global destructive pathogens including rice stripe virus, Magnaporthe oryzae, and Xanthomonas oryzae pv. oryzae.

Prevalence of Vector-Borne Pathogens in Reproductive and Non-Reproductive Tissue Samples from Free-Roaming Domestic Cats in the South Atlantic USA.

Reservoir to multiple species of zoonotic pathogens, free-roaming cats (FRCs) interact with domestic and wild animals, vectors, and humans. To assess the potential for feline vector-borne pathogens to be vertically transmitted, this study surveyed ear tip and reproductive tissues of FRCs from two locations in the South Atlantic United States for Anaplasma, Bartonella, Ehrlichia, hemotropic Mycoplasma, and Rickettsia species. We collected ovary (n = 72), uterus (n = 54), testicle (n = 74), and ear tip (n = 73) tissue from 73 cats, and fetal (n = 20) and placental (n = 19) tissue from 11 queens. Pathogen DNA was amplified utilizing qPCR, confirmed by sequencing. Cats were more frequently Bartonella henselae positive on reproductive tissues (19%, 14/73) than ear tip (5%, 4/73; p = 0.02). B. henselae was amplified from fetus (20%, 4/20) and placenta samples (11%, 2/19). Bartonella spp. infection was more common in cats from North Carolina (76%, 26/34) than Virginia (13%, 5/39; p < 0.0001). Fourteen percent (10/73) of both ear tip and reproductive tissues were positive for hemotropic Mycoplasma spp. Anaplasma, Ehrlichia, and Rickettsia spp. DNA was not amplified from any cat/tissue. These findings suggest that B. henselae preferentially infected cats’ reproductive tissue and reinforces the importance of investigating the potential for B. henselae vertical transmission or induction of reproductive failure.

Detection of Multiple Intracellular Bacterial Pathogens in Haemaphysalis flava Ticks Collected from Hedgehogs in Central China.

Tickborne intracellular bacterial pathogens including Anaplasma, Coxiella burnetti, Ehrlichia, and Rickettsia cause emerging infectious diseases worldwide. PCR was used to amplify the genes of these pathogens in Haemaphysalis flava ticks collected from hedgehogs in Central China. Among 125 samples including 20 egg batches, 24 engorged females, and 81 molted male and female adult ticks, the DNA sequences and phylogenetic analysis showed that the minimum infection rate of the ticks was 4% (5/125) for A. bovis, 3.2% (4/125) for C. burnetti, 9.6%, (12/125) for E. ewingii, and 5.6% for Rickettsia including R. japonica (3.2%, 4/125) and R. raoultii (2.4%, 3/125), respectively. The prevalence of these pathogens was significantly higher in dead engorged females (83.3%, 20/24) than in eggs (5%, 1/20) and molted ticks (8.6%, 7/81). Our study indicated that H. flava ticks could be infected with multiple species of tickborne pathogens including Anaplasma, C. burnetti, Ehrlichia, and Rickettsia in Central China, and the prevalence of these pathogens was reduced during transovarial and transstadial transmission in ticks, suggesting that ticks may not be real reservoirs but only vectors for these tickborne pathogens.

An Erg11 lanosterol 14-α-demethylase-Arv1 complex is required for Candida albicans virulence.

Azole resistant fungal infections remain a health problem for the immune compromised. Current therapies are limited due to rises in new resistance mechanisms. Therefore, it is important to identify new drug targets for drug discovery and novel therapeutics. Arv1 (are1 are2 required for viability 1) function is highly conserved between multiple pathogenic fungal species. Candida albicans (C. albicans) cells lacking CaArv1 are azole hypersusceptible and lack virulence. Saccharomyces cerevisiae (S. cerevisiae) Scarv1 cells are also azole hypersusceptible, a phenotype reversed by expression of CaArv1, indicating conservation in the molecular mechanism for azole susceptibility. To define the relationship between Arv1 function and azole susceptibility, we undertook a structure/function analysis of ScArv1. We identified several conserved amino acids within the ScArv1 homology domain (ScAhd) required for maintaining normal azole susceptibility. Erg11 lanosterol 14-α-demethylase is the rate-limiting enzyme in sterol biosynthesis and is the direct target of azole antifungals, so we used our ScArv1 mutants in order to explore the relationship between ScArv1 and ScErg11. Specific ScArv1 mutants ectopically expressed from a low copy plasmid were unable to restore normal azole susceptibility to Scarv1 cells and had reduced Erg11 protein levels. Erg11 protein stability depended on its ability to form a heterodimeric complex with Arv1. Complex formation was required for maintaining normal azole susceptibility. Scarv1 cells expressing orthologous CaArv1 mutants also had reduced CaErg11 levels, were unable to form a CaArv1-CaErg11 complex, and were azole hypersusceptible. Scarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 could not sustain proper levels of the azole resistant CaErg11Y132F F145L protein. Caarv1/Caarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 were found to lack virulence using a disseminated candidiasis mouse model. Expressing CaErg11Y132F F145L did not reverse the lack of virulence. We hypothesize that the role of Arv1 in Erg11-dependent azole resistance is to stabilize Erg11 protein level. Arv1 inhibition may represent an avenue for treating azole resistance.