Search

The genetic diversity of pathogens, and interactions between genotypes, can strongly influence pathogen phenotypes such as transmissibility and virulence. For vector-borne pathogens, both mammalian hosts and arthropod vectors may limit pathogen genotypic diversity (number of unique genotypes circulating in an area) by preventing infection or transmission of particular genotypes. Mammalian hosts often act as “ecological filters” for pathogen diversity, where novel variants are frequently eliminated because of stochastic events or fitness costs. However, whether vectors can serve a similar role in limiting pathogen diversity is less clear. Here we show using Francisella novicida and a natural tick vector of Francisella spp. (Dermacentor andersoni), that the tick vector acted as a stronger ecological filter for pathogen diversity compared to the mammalian host. When both mice and ticks were exposed to mixtures of F. novicida genotypes, significantly fewer genotypes co-colonized ticks compared to mice. In both ticks and mice, increased genotypic diversity negatively affected the recovery of available genotypes. Competition among genotypes contributed to the reduction of diversity during infection of the tick midgut, as genotypes not recovered from tick midguts during mixed genotype infections were recovered from tick midguts during individual genotype infection. Mediated by stochastic and selective forces, pathogen genotype diversity was markedly reduced in the tick. We incorporated our experimental results into a model to demonstrate how vector population dynamics, especially vector-to-host ratio, strongly affected pathogen genotypic diversity in a population over time. Understanding pathogen genotypic population dynamics will aid in identification of the variables that most strongly affect pathogen transmission and disease ecology., Author Summary Co-infection, the presence of multiple genotypes of the same pathogen species within an infected individual, is common. Genotype diversity, defined as the number of unique genotypes, and the interaction between genotypes, can strongly influence virulence and pathogen transmission. Understanding how genotypic diversity affects transmission of pathogens that naturally cycle among disparate hosts, such as vector-borne pathogens, is especially important as the capacity of the host and vector to sustain genotypic diversity may differ. To address this, we exposed Dermacentor andersoni ticks, via infected mice, to variably diverse populations of Francisella novicida genotypes. Interestingly, we found that ticks served as greater ecological filters for genotypic diversity compared to mice. This loss in genotypic diversity was due to both stochastic and selective forces. Based on these data and a model, we determined that high numbers of ticks in an environment support high genotypic diversity, while genotypic diversity will be lost rapidly in environments with low tick numbers. Together, these results provide evidence that vector population dynamics, vector-to-host ratios, and competition among pathogen genotypes play critical roles in the maintenance of pathogen genotypic diversity.

Paenibacillus larvae, the etiological agent of the globally occurring epizootic American Foulbrood (AFB) of honey bees, causes intestinal infections in honey bee larvae which develop into systemic infections inevitably leading to larval death. Massive brood mortality might eventually lead to collapse of the entire colony. Molecular mechanisms of host-microbe interactions in this system and of differences in virulence between P. larvae genotypes are poorly understood. Recently, it was demonstrated that the degradation of the peritrophic matrix lining the midgut epithelium is a key step in pathogenesis of P. larvae infections. Here, we present the isolation and identification of PlCBP49, a modular, chitin-degrading protein of P. larvae and demonstrate that this enzyme is crucial for the degradation of the larval peritrophic matrix during infection. PlCBP49 contains a module belonging to the auxiliary activity 10 (AA10, formerly CBM33) family of lytic polysaccharide monooxygenases (LPMOs) which are able to degrade recalcitrant polysaccharides. Using chitin-affinity purified PlCBP49, we provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for members of the AA10 family. Using P. larvae mutants lacking PlCBP49 expression, we analyzed in vivo biological functions of PlCBP49. In the absence of PlCBP49 expression, peritrophic matrix degradation was markedly reduced and P. larvae virulence was nearly abolished. This indicated that PlCBP49 is a key virulence factor for the species P. larvae. The identification of the functional role of PlCBP49 in AFB pathogenesis broadens our understanding of this important family of chitin-binding and -degrading proteins, especially in those bacteria that can also act as entomopathogens., Author Summary American Foulbrood and its etiological agent, Paenibacillus larvae, pose a serious threat to global honey bee health. So far, molecular mechanisms of host-microbe interactions are poorly understood in this system and no key virulence factor for the entire species has been identified. Here, we demonstrate that P. larvae expresses a chitin-binding and -degrading protein PlCBP49 harboring one module that belongs to the auxiliary activity 10 (AA10) family of lytic polysaccharide monooxygenases (LPMOs). We provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for other members of the AA10 enzyme family. Using P. larvae mutants lacking PlCBP49 expression, we demonstrate that PlCBP49 is crucial for the degradation of the chitin-rich peritrophic matrix, a key step in pathogenesis of P. larvae infections. In the absence of PlCBP49 expression the peritrophic matrix remained nearly intact and about 95% of the infected larvae survived infection. This clearly indicated that PlCBP49 is a key virulence factor of P. larvae. These results constitute important progress in our understanding of both P. larvae pathogenesis and the biological role of LPMOs in entomopathogens. Furthermore, knowing PlCBP49 and its role in pathogenesis opens new possibilities to develop curative measures for this disease.

Atypical porcine reproductive and respiratory syndrome (PRRS), which is caused by the Chinese highly pathogenic PRRS virus (HP-PRRSV), has resulted in large economic loss to the swine industry since its outbreak in 2006. However, to date, the region(s) within the viral genome that are related to the fatal virulence of HP-PRRSV remain unknown. In the present study, we generated a series of full-length infectious cDNA clones with swapped coding regions between the highly pathogenic RvJXwn and low pathogenic RvHB-1/3.9. Next, the in vitro and in vivo replication and pathogenicity for piglets of the rescued chimeric viruses were systematically analyzed and compared with their backbone viruses. First, we swapped the regions including the 5′UTR+ORF1a, ORF1b, and structural proteins (SPs)-coding region between the two viruses and demonstrated that the nonstructural protein-coding region, ORF1b, is directly related to the fatal virulence and increased replication efficiency of HP-PRRSV both in vitro and in vivo. Furthermore, we substituted the nonstructural protein (Nsp) 9-, Nsp10-, Nsp11- and Nsp12-coding regions separately; or Nsp9- and Nsp10-coding regions together; or Nsp9-, Nsp10- and Nsp11-coding regions simultaneously between the two viruses. Our results indicated that the HP-PRRSV Nsp9- and Nsp10-coding regions together are closely related to the replication efficiency in vitro and in vivo and are related to the increased pathogenicity and fatal virulence for piglets. Our findings suggest that Nsp9 and Nsp10 together contribute to the fatal virulence of HP-PRRSV emerging in China, helping to elucidate the pathogenesis of this virus., Author Summary PRRS is a considerable threat to the pig industry worldwide. A large-scale atypical PRRS caused by highly pathogenic PRRSV (HP-PRRSV) that emerged in 2006 has resulted in considerable economic loss to Chinese pig production. The disease is characterized by a high body temperature (41°C–42°C), morbidity and by mortality of the affected pigs. Although the genomic marker, the 30-amino-acid deletion in its Nsp2-coding region has been previously verified to have no relation to its increased pathogenicity, the genomic region(s) associated with the fatal virulence of HP-PRRSV remain unclear. A series of chimeric viruses with swapped coding regions between HP- and LP-PRRSV were constructed, and their growth abilities and pathogenicities in piglets were analyzed. Our results demonstrated that Nsp9 and Nsp10 together contribute to the replication efficiency and the fatal virulence of HP-PRRSV for piglets. Our finding is not only the first unambiguous illumination concerning the key virulence determinant of Chinese HP-PRRSV but it also provides a novel insight for understanding the molecular pathogenesis of this virus and for designing new drugs and vaccines against PRRSV infection in the future.

Sapovirus, a member of the Caliciviridae family, is an important cause of acute gastroenteritis in humans and pigs. Currently, the porcine sapovirus (PSaV) Cowden strain remains the only cultivable member of the Sapovirus genus. While some caliciviruses are known to utilize carbohydrate receptors for entry and infection, a functional receptor for sapovirus is unknown. To characterize the functional receptor of the Cowden strain of PSaV, we undertook a comprehensive series of protein-ligand biochemical assays in mock and PSaV-infected cell culture and/or piglet intestinal tissue sections. PSaV revealed neither hemagglutination activity with red blood cells from any species nor binding activity to synthetic histo-blood group antigens, indicating that PSaV does not use histo-blood group antigens as receptors. Attachment and infection of PSaV were markedly blocked by sialic acid and Vibrio cholerae neuraminidase (NA), suggesting a role for α2,3-linked, α2,6-linked or α2,8-linked sialic acid in virus attachment. However, viral attachment and infection were only partially inhibited by treatment of cells with sialidase S (SS) or Maackia amurensis lectin (MAL), both specific for α2,3-linked sialic acid, or Sambucus nigra lectin (SNL), specific for α2,6-linked sialic acid. These results indicated that PSaV recognizes both α2,3- and α2,6-linked sialic acids for viral attachment and infection. Treatment of cells with proteases or with benzyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside (benzylGalNAc), which inhibits O-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or N-linked glycosylation had no such effect on virus binding or infection. These data suggest PSaV binds to cellular receptors that consist of α2,3- and α2,6-linked sialic acids on glycoproteins attached via O-linked glycosylation., Author Summary Although enteropathogenic sapoviruses and noroviruses are leading causes of acute gastroenteritis in both humans and animals, the study of viral pathogenesis and immunity of these ubiquitous pathogens has been hampered due to the lack of a fully permissive cell culture system. Porcine sapovirus Cowden strain provides a suitable system that can be used to identify the molecular mechanisms of viral pathogenesis. Previous studies have shown that carbohydrates and glycolipids play important roles in the attachment of members of the Caliciviridae; histo-blood group antigens (HBGAs) are used by Norovirus genogroups I to IV, as well as members of the Lagovirus, and Recovirus genera, whereas terminal sialic acid is recognized as a receptor for feline calicivirus and murine norovirus. To date, however, the role of carbohydrates in the life cycle of sapoviruses has remained largely unknown. We found that porcine sapovirus binds to susceptible host cells through both α2,3- and α2,6-linked terminal sialic acids which are attached to O-linked glycoproteins. These efforts, findings and insights will significantly contribute to a better understanding of the sapovirus life cycle.

Over 1,000,000,000 people and innumerable other animals are currently infected with one or more helminths [1]. These highly prevalent infections contribute to significant illness and economic losses due to impaired worker productivity and livestock health. As such, it is tremendously important to vaccine and anthelminthic drug development efforts to understand the complex interactions between host and parasite, including helminth manipulation of host tissues. The extensive coevolution of helminths and their hosts often blurs the line between what is host-mediated and what is parasite-driven. However, we have drawn explicit examples from the literature to highlight five major themes of helminth manipulation of host tissues: disruption of epithelial barriers, reinforcement of epithelial barriers, alteration of lymphoid tissue, modulation of tissue vascularity, and tuning of granulomatous responses. Herein we focus on examples of direct molecular level effects on host tissue to highlight strategies common to numerous helminths in their efforts to survive and reproduce within their hosts.

Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with >10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil's) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of “core” housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaption, selection of targets for mutagenesis, and the identification of previously unrecognized virulence determinants., Author Summary Leptospirosis, a global disease caused by the unusual bacterium Leptospira, is transmitted from animals to humans. Pathogenic species of Leptospira are excreted in urine from infected animals and can continue to survive in suitable environments before coming into contact with a new reservoir or accidental host. Leptospires have an inherent ability to survive a wide range of conditions encountered in nature during transmission and within mammals. However, we know very little about the regulatory pathways and gene products that promote mammalian host adaptation and enable leptospires to establish infection. In this study, we used a novel system whereby leptospires are cultivated in dialysis membrane chambers implanted into the peritoneal cavities of rats to compare the gene expression profiles of mammalian host-adapted and in vitro-cultivated organisms. In addition to providing a facile system for studying the transcriptional and physiologic changes leptospires undergo during mammalian infection, our data provide a rational basis for selecting new targets for mutagenesis.

1 Comparative Immunogenetics Laboratory, Texas A&M University, College Station, Texas, United States of America, 2 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States of America, 3 Department of Plant Pathology and Microbiology, College of Agriculture and Life Sciences, Texas A&M University, College Station, Texas, United States of America, 4 Norman Borlaug Center, Texas A&M University, College Station, Texas, United States of America, 5 Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center, College Station, Texas, United States of America

Low-cost DNA sequencing has greatly democratized genomics, especially for the typically very small genomes of viruses [1]–[3]. The recent acceleration in human virus discovery by metagenomics indicates that many viruses escaped prior detection due to limitations of preexisting technologies. It is now conceivable that all viral species commonly infecting human (i.e., the human virome) will soon be determined. As the value of the human genome and microbiome has become widely recognized, providing crucial reference genomes and opening unanticipated avenues of research, the genetic characterization of the human virome also holds great promises [4]. The human microbiome project, focusing largely on single bacterial cells and metagenomic sequencing of total DNA from feces and other human sites, is unlikely to detect RNA viruses. The generally minuscule size of viral genomes relative to those of their bacterial or eukaryotic hosts also weighs against their easy detection in metagenomic approaches. Viral discovery can be greatly facilitated by simple filtration to enrich the smaller viral particles and by removal of contaminating bacterial and human nucleic acids using nuclease digestion that leave viral nucleic acids protected within their virion shells (Figure 1) [5]. Density gradient ultra-centrifugation has also been used to enrich viral particles. Figure 1 Schematic steps for determination of human virome and its impact on health, including biological samples and data acquisition, genetic analysis, and epidemiology of viral infections. Knowledge of the human virome will allow the design of sensitive and specific tests to all human viruses using “virochip” microarrays [6] or multiplexed PCR assays (Figure 1). Provided samples from large enough human cohorts are analyzed, it will be possible to determine viral prevalence, likely transmission routes, and most crucially disease association. The development of vaccines and antivirals can then be targeted to the viruses with the largest public health impacts in different geographic regions. Deciphering the complete human virome will therefore improve our understanding, prevention, and treatment of currently unexplained diseases involving viral pathogens, as well as provide insights into the evolution of viral families and cross-species viral transmissions. Despite the rapid progress being made toward deciphering the human virome, several roadblocks remain to its full characterization and utilization. Below is an abbreviated list of these problems and possible solutions.

Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex group of bacteria. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB. Identification of biomarkers in tuberculosis research remains elusive and the goal is to identify host correlates of protection. We hypothesized that by studying global gene expression we could identify in vitro predictors of protection that could help to facilitate vaccine development. Calves were vaccinated with BCG or with a heterologous BCG prime adenovirally vectored subunit boosting protocol. Protective efficacy was determined after M. bovis challenge. RNA was prepared from PPD-stimulated PBMC prepared from vaccinated-protected, vaccinated-unprotected and unvaccinated control cattle prior to M. bovis challenge and global gene expression determined by RNA-seq. 668 genes were differentially expressed in vaccinated-protected cattle compared with vaccinated-unprotected and unvaccinated control cattle. Cytokine-cytokine receptor interaction was the most significant pathway related to this dataset with IL-22 expression identified as the dominant surrogate of protection besides INF-γ. Finally, the expression of these candidate genes identified by RNA-seq was evaluated by RT-qPCR in an independent set of PBMC samples from BCG vaccinated and unvaccinated calves. This experiment confirmed the importance of IL-22 as predictor of vaccine efficacy., Author Summary Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of a complex group of pathogens that also includes the bacterium causing human tuberculosis, M. tuberculosis. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB. In addition to the relevance for the veterinary field, cattle can also serve as useful pre-clinical model for the development of human vaccines. Identification of biomarkers in tuberculosis research remains elusive and the goal of this study was to identify host markers that allow the prediction of vaccine success. In this experiment, the outcome of vaccination was measured against relevant endpoints based on disease severity. Our results, generated using the state-of-the-art methodology of transcriptome sequencing defined a dominant role of the cytokine IL-22 for predicting vaccine success. We therefore identified a predictive biomarker that can be used in future experiments and also highlighted the role of T cells producing this cytokine in protective immunity.

Idiopathic chronic diarrhea (ICD) is a leading cause of morbidity amongst rhesus monkeys kept in captivity. Here, we show that exposure of affected animals to the whipworm Trichuris trichiura led to clinical improvement in fecal consistency, accompanied by weight gain, in four out of the five treated monkeys. By flow cytometry analysis of pinch biopsies collected during colonoscopies before and after treatment, we found an induction of a mucosal TH2 response following helminth treatment that was associated with a decrease in activated CD4+ Ki67+ cells. In parallel, expression profiling with oligonucleotide microarrays and real-time PCR analysis revealed reductions in TH1-type inflammatory gene expression and increased expression of genes associated with IgE signaling, mast cell activation, eosinophil recruitment, alternative activation of macrophages, and worm expulsion. By quantifying bacterial 16S rRNA in pinch biopsies using real-time PCR analysis, we found reduced bacterial attachment to the intestinal mucosa post-treatment. Finally, deep sequencing of bacterial 16S rRNA revealed changes to the composition of microbial communities attached to the intestinal mucosa following helminth treatment. Thus, the genus Streptophyta of the phylum Cyanobacteria was vastly increased in abundance in three out of five ICD monkeys relative to healthy controls, but was reduced to control levels post-treatment; by contrast, the phylum Tenericutes was expanded post-treatment. These findings suggest that helminth treatment in primates can ameliorate colitis by restoring mucosal barrier functions and reducing overall bacterial attachment, and also by altering the communities of attached bacteria. These results also define ICD in monkeys as a tractable preclinical model for ulcerative colitis in which these effects can be further investigated., Author Summary Young macaques kept in captivity at Primate Research Centers often develop chronic diarrhea, which is difficult to treat because it is poorly understood. This disease shares many features with ulcerative colitis, which is an autoimmune disease affecting the intestinal tract of humans. Recently, parasitic worms have been used in clinical trials to treat inflammatory bowel diseases in humans with positive results, but very little is known about how worms can improve symptoms. We performed a trial where we treated macaques suffering from chronic diarrhea with human whipworms, collecting gut biopsies before and after treatment. We found that 4 out of the 5 treated macaques improved their symptoms and studied the changes in their gut immune responses, as they got better. We found that after treatment with worms, the monkeys had less bacteria attached to their intestinal wall and a reduced inflammatory response to the gut bacteria. Additionally, the composition of gut bacteria was altered in the sick macaques and was restored close to normal after treatment with whipworms. These results provide a potential mechanism by which parasitic worms may improve the symptoms of intestinal inflammation, by reducing the immune response against intestinal bacteria.

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins., Author Summary Many significant infectious diseases that impact human health evolve in animal hosts. Our work focuses on infections caused by strains of enterohemorrhagic Escherichia coli (EHEC) that cause bloody diarrhoea and life threatening kidney and brain damage in humans as an incidental host, while ruminants are a reservoir host. EHEC strains are infected with bacteriophages that can integrate their genetic material into the bacterial chromosome. This includes genes for the production of Shiga toxins (Stx) that are responsible for the severe pathology in humans. It has been demonstrated that certain EHEC strains are more likely to be associated with human disease and ‘supershedding’ animals. The current study has shown that these EHEC strains are more likely to contain two related Stx bacteriophages, rather than one, and that the intercalating bacteriophages take control of the bacterial type III secretion system that is essential for ruminant colonization. We propose that this regulation favours co-acquisition of other genetic regions that encode type III-secreted proteins and regulators that can overcome this control. This finding helps our understanding of EHEC strain evolution and indicates that selection of more toxic strains may be occurring in the ruminant host with important implications for human health.

The cell intrinsic innate immune responses provide a first line of defense against viral infection, and often function by targeting cellular pathways usurped by the virus during infection. In particular, many viruses manipulate cellular lipids to form complex structures required for viral replication, many of which are dependent on de novo fatty acid synthesis. We found that the energy regulator AMPK, which potently inhibits fatty acid synthesis, restricts infection of the Bunyavirus, Rift Valley Fever Virus (RVFV), an important re-emerging arthropod-borne human pathogen for which there are no effective vaccines or therapeutics. We show restriction of RVFV both by AMPK and its upstream activator LKB1, indicating an antiviral role for this signaling pathway. Furthermore, we found that AMPK is activated during RVFV infection, leading to the phosphorylation and inhibition of acetyl-CoA carboxylase, the first rate-limiting enzyme in fatty acid synthesis. Activating AMPK pharmacologically both restricted infection and reduced lipid levels. This restriction could be bypassed by treatment with the fatty acid palmitate, demonstrating that AMPK restricts RVFV infection through its inhibition of fatty acid biosynthesis. Lastly, we found that this pathway plays a broad role in antiviral defense since additional viruses from disparate families were also restricted by AMPK and LKB1. Therefore, AMPK is an important component of the cell intrinsic immune response that restricts infection through a novel mechanism involving the inhibition of fatty acid metabolism., Author Summary RNA viruses represent an important worldwide source of infection and disease in both humans and animals. While individual viruses have unique characteristics, some stages of infection are conserved and must be completed in order to successfully infect and grow. Viruses must undergo genome replication, protein synthesis, and assembly of new virus particles. In particular, numerous RNA viruses manipulate cellular membranes to create new complex structures required for viral replication in a process that is often dependent on fatty acid biosynthesis. This is a process that is tightly regulated by the energy sensor AMPK. We have found that energy-mediated activation of AMPK restricts infection of the Bunyavirus Rift Valley fever virus by decreasing levels of fatty acid synthesis. Furthermore, several additional RNA viruses from disparate families that share this dependence of membrane modification and fatty acid synthesis are also restricted by AMPK. Thus AMPK likely represents a novel component of the cell intrinsic immune response to RNA viruses, and may be a good target for the development of antiviral therapeutics against a range of medically important viruses.

Two recently emerged cutaneous fungal diseases of wildlife, bat white-nose syndrome (WNS) [1] and amphibian chytridiomycosis [2], have devastated affected populations. Fungal diseases are gaining recognition as significant causes of morbidity and mortality to plants, animals, and humans [3], yet fewer than 10% of fungal species are known [4]. Furthermore, limited antifungal therapeutic drugs are available, antifungal therapeutics often have associated toxicity, and there are no approved antifungal vaccines. The unexpected emergence of WNS, the rapidity with which it has spread, and its unprecedented severity demonstrate both the impacts of novel fungal disease upon naive host populations and challenges to effective management of such diseases.

The Gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a globally occurring, deathly epizootic of honey bee brood. AFB outbreaks are predominantly caused by two genotypes of P. larvae, ERIC I and ERIC II, with P. larvae ERIC II being the more virulent genotype on larval level. Recently, comparative proteome analyses have revealed that P. larvae ERIC II but not ERIC I might harbour a functional S-layer protein, named SplA. We here determine the genomic sequence of splA in both genotypes and demonstrate by in vitro self-assembly studies of recombinant and purified SplA protein in combination with electron-microscopy that SplA is a true S-layer protein self-assembling into a square 2D lattice. The existence of a functional S-layer protein is novel for this bacterial species. For elucidating the biological function of P. larvae SplA, a genetic system for disruption of gene expression in this important honey bee pathogen was developed. Subsequent analyses of in vivo biological functions of SplA were based on comparing a wild-type strain of P. larvae ERIC II with the newly constructed splA-knockout mutant of this strain. Differences in cell and colony morphology suggest that SplA is a shape-determining factor. Marked differences between P. larvae ERIC II wild-type and mutant cells with regard to (i) adhesion to primary pupal midgut cells and (ii) larval mortality as measured in exposure bioassays corroborate the assumption that the S-layer of P. larvae ERIC II is an important virulence factor. Since SplA is the first functionally proven virulence factor for this species, our data extend the knowledge of the molecular differences between these two genotypes of P. larvae and contribute to explaining the observed differences in virulence. These results present an immense advancement in our understanding of P. larvae pathogenesis., Author Summary Paenibacillus larvae is the most devastating bacterial pathogen of honey bees. However, the molecular interactions between infected larvae and P. larvae are poorly understood and little more than speculation exist concerning virulence factors. Recently, a putative S-layer protein has been identified in P. larvae. We here demonstrate that only representatives of P. larvae genotype ERIC II harbor a functional splA-gene and that SplA is a true S-layer protein with self-assembly capability. The presence of a functional S-layer protein is novel for P. larvae. When elucidating the biological function of SplA we broke new ground by establishing primary cell culture for pupal gut cells and by developing a genetic system for disruption of gene expression in this important honey bee pathogen. By using these novel methods we were able to prove that SplA serves as a shape-determining factor, mediates adhesion to host cells, and is a key virulence factor of P. larvae ERIC II. These results present an immense advancement in our understanding of P. larvae pathogenesis. Furthermore, we propose P. larvae as a model system for the analysis of the in vivo functions of S-layer proteins because P. larvae SlpA knockout-mutants retain viability and are thus suitable for functional studies.

Two-cysteine peroxiredoxins are ubiquitous peroxidases that play various functions in cells. In Leishmania and related trypanosomatids, which lack catalase and selenium-glutathione peroxidases, the discovery of this family of enzymes provided the molecular basis for peroxide removal in these organisms. In this report the functional relevance of one of such enzymes, the mitochondrial 2-Cys peroxiredoxin (mTXNPx), was investigated along the Leishmania infantum life cycle. mTXNPx null mutants (mtxnpx−) produced by a gene replacement strategy, while indistinguishable from wild type promastigotes, were found unable to thrive in a murine model of infection. Unexpectedly, however, the avirulent phenotype of mtxnpx− was not due to lack of the peroxidase activity of mTXNPx as these behaved like controls when exposed to oxidants added exogenously or generated by macrophages during phagocytosis ex vivo. In line with this, mtxnpx− were also avirulent when inoculated into murine hosts unable to mount an effective oxidative phagocyte response (B6.p47phox−/− and B6.RAG2−/− IFN-γ−/− mice). Definitive conclusion that the peroxidase activity of mTXNPx is not required for parasite survival in mice was obtained by showing that a peroxidase-inactive version of this protein was competent in rescuing the non-infective phenotype of mtxnpx−. A novel function is thus proposed for mTXNPx, that of a molecular chaperone, which may explain the impaired infectivity of the null mutants. This premise is based on the observation that the enzyme is able to suppress the thermal aggregation of citrate synthase in vitro. Also, mtxnpx− were more sensitive than controls to a temperature shift from 25°C to 37°C, a phenotype reminiscent of organisms lacking specific chaperone genes. Collectively, the findings reported here change the paradigm which regards all trypanosomatid 2-Cys peroxiredoxins as peroxide-eliminating devices. Moreover, they demonstrate, for the first time, that these 2-Cys peroxiredoxins can be determinant for pathogenicity independently of their peroxidase activity., Author Summary Leishmania are insect-borne protozoan parasites that cause leishmaniasis, a spectrum of diseases with relevance in men and dogs and for which there are no adequate means of control. In order to successfully infect their mammalian hosts, Leishmania requires the expression of a set of key proteins. Here, we identify one mitochondrial protein of the parasite as one of such molecules. Interestingly, however, we also discovered that the reason why this enzyme is essential is not due to its well characterized peroxidase activity, which defines it as a member of the peroxiredoxin family. Instead, the importance of this protein during infection may derive from its ability to function as a chaperone, as implied here. In short, by proposing a novel, peroxidase-unrelated role for the Leishmania mitochondrial peroxiredoxin during mammals' infection, this work opens new perspectives regarding the physiologic function of this enzyme.

Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551), ipdB (rv3552), fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and β-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD) and 3aα-H-4α(3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP). Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections., Author Summary Rhodococcus equi causes fatal pyogranulomatous bronchopneumonia in young foals and is an emerging opportunistic pathogen of immunocompromised humans. Despite its importance, there is currently no safe and effective vaccine against R. equi infections. Like Mycobacterium tuberculosis, the causative agent of human tuberculosis, R. equi is able to infect, survive and multiply inside alveolar macrophages. Recently we have shown that essential steps in the cholesterol catabolic pathway (encoded by the rv3551, rv3552, fadE30 genes) are involved in the pathogenicity of M. tuberculosis. We hypothesized that the orthologous genes in the cholesterol catabolic gene cluster of R. equi also are essential for its virulence mechanism. Analysis of the respective R. equi strain RE1 mutants revealed that they were impaired in growth on intermediates of the steroid catabolic pathway and had attenuated phenotypes in a macrophage infection assay. Mutant RE1ΔipdAB, carrying a deletion of the orthologs of rv3551 and rv3552, could be safely administered to 2–5 week-old foals intratracheally and oral immunization provided a substantial protection against infection by a virulent R. equi strain. Our data show that genes important for methylhexahydroindanone propionate degradation, part of the steroid catabolic pathway, are promising targets for the development of a live-attenuated vaccine against R. equi infections.

Rabbit Hemorrhagic disease virus (RHDV), a calicivirus of the Lagovirus genus, and responsible for rabbit hemorrhagic disease (RHD), kills rabbits between 48 to 72 hours post infection with mortality rates as high as 50–90%. Caliciviruses, including noroviruses and RHDV, have been shown to bind histo-blood group antigens (HBGA) and human non-secretor individuals lacking ABH antigens in epithelia have been found to be resistant to norovirus infection. RHDV virus-like particles have previously been shown to bind the H type 2 and A antigens. In this study we present a comprehensive assessment of the strain-specific binding patterns of different RHDV isolates to HBGAs. We characterized the HBGA expression in the duodenum of wild and domestic rabbits by mass spectrometry and relative quantification of A, B and H type 2 expression. A detailed binding analysis of a range of RHDV strains, to synthetic sugars and human red blood cells, as well as to rabbit duodenum, a likely gastrointestinal site for viral entrance was performed. Enzymatic cleavage of HBGA epitopes confirmed binding specificity. Binding was observed to blood group B, A and H type 2 epitopes in a strain-dependent manner with slight differences in specificity for A, B or H epitopes allowing RHDV strains to preferentially recognize different subgroups of animals. Strains related to the earliest described RHDV outbreak were not able to bind A, whereas all other genotypes have acquired A binding. In an experimental infection study, rabbits lacking the correct HBGA ligands were resistant to lethal RHDV infection at low challenge doses. Similarly, survivors of outbreaks in wild populations showed increased frequency of weak binding phenotypes, indicating selection for host resistance depending on the strain circulating in the population. HBGAs thus act as attachment factors facilitating infection, while their polymorphism of expression could contribute to generate genetic resistance to RHDV at the population level., Author Summary Rabbit hemorrhagic disease virus (RHDV), detected as late as 1984, has spread to large parts of the world, threatening rabbit populations and other species dependent on rabbits in many European countries. Mortality has been shown to be as high as 90% and rabbits are killed 48 to 72 hours after infection. Related viruses called noroviruses, infect humans in a manner dependent on the expression of histo-blood group antigens (HBGAs), which are not only expressed on red blood cells, but also on epithelial cells, in saliva and on mucins of the intestinal tract. RHDV also binds to HBGA and in this report we characterize binding of strains of all genetic groups of RHDV to different HBGAs. We also demonstrate HBGAs to function as attachment factors in a challenge experiment. As polymorphisms of genes involved in HBGA synthesis divide the rabbit population into different subgroups, we find selection of low-binding subgroups of wild rabbits in populations recovering from devastating outbreaks of RHDV. This is the first demonstration of differential HBGA specificities of RHDV strains, description of function in infection and demonstration of host selection due to RHDV infection based on HBGA phenotype.

Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, Brucella abortus is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-γ and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular Brucella infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity., Author Summary In some diseases the predominant cells recruited are PMNs while in others are mononuclear leukocytes. Traditionally, this marked the difference between acute and chronic infections, a perspective reinforced by in vivo models in which immune cells are depleted by means of antibodies. However, these models have several drawbacks and knock-out mice were generated to dissect the functionality of immune cells. Despite this, the study of PMNs in infections in which adaptive immunity plays a role has been precluded by the absence of long-lasting neutropenic models. A mouse strain named Genista, in which the defect is the absence of PMNs has been developed; thus, making possible to explore the role of PMNs during adaptive immunity in chronic infections. We have used Brucella, an intracellular pathogen that avoids degranulation and stands the killing action of PMNs. Instead, Brucella causes chronicity, inducing granulomas, recruitment of macrophages/DCs and a robust adaptive immune response. We found that the absence PMNs is non-lethal and favors Brucella elimination at later times of infection, a phenomenon that correlates with the balance of Th1 over Th2 response. We propose that beside their role in primary bacterial elimination, PMNs can dampen and participate in regulatory circuits of adaptive immune response.

Summary: Explaining the contribution of host and pathogen factors in driving infection dynamics is a major ambition in parasitology. There is increasing recognition that analyses based on single summary measures of an infection (e.g., peak parasitaemia) do not adequately capture infection dynamics and so, the appropriate use of statistical techniques to analyse dynamics is necessary to understand infections and, ultimately, control parasites. However, the complexities of within-host environments mean that tracking and analysing pathogen dynamics within infections and among hosts poses considerable statistical challenges. Simple statistical models make assumptions that will rarely be satisfied in data collected on host and parasite parameters. In particular, model residuals (unexplained variance in the data) should not be correlated in time or space. Here we demonstrate how failure to account for such correlations can result in incorrect biological inference from statistical analysis. We then show how mixed effects models can be used as a powerful tool to analyse such repeated measures data in the hope that this will encourage better statistical practices in parasitology.

The frequent interactions of rodents with humans make them a common source of zoonotic infections. To obtain an initial unbiased measure of the viral diversity in the enteric tract of wild rodents we sequenced partially purified, randomly amplified viral RNA and DNA in the feces of 105 wild rodents (mouse, vole, and rat) collected in California and Virginia. We identified in decreasing frequency sequences related to the mammalian viruses families Circoviridae, Picobirnaviridae, Picornaviridae, Astroviridae, Parvoviridae, Papillomaviridae, Adenoviridae, and Coronaviridae. Seventeen small circular DNA genomes containing one or two replicase genes distantly related to the Circoviridae representing several potentially new viral families were characterized. In the Picornaviridae family two new candidate genera as well as a close genetic relative of the human pathogen Aichi virus were characterized. Fragments of the first mouse sapelovirus and picobirnaviruses were identified and the first murine astrovirus genome was characterized. A mouse papillomavirus genome and fragments of a novel adenovirus and adenovirus-associated virus were also sequenced. The next largest fraction of the rodent fecal virome was related to insect viruses of the Densoviridae, Iridoviridae, Polydnaviridae, Dicistroviriade, Bromoviridae, and Virgaviridae families followed by plant virus-related sequences in the Nanoviridae, Geminiviridae, Phycodnaviridae, Secoviridae, Partitiviridae, Tymoviridae, Alphaflexiviridae, and Tombusviridae families reflecting the largely insect and plant rodent diet. Phylogenetic analyses of full and partial viral genomes therefore revealed many previously unreported viral species, genera, and families. The close genetic similarities noted between some rodent and human viruses might reflect past zoonoses. This study increases our understanding of the viral diversity in wild rodents and highlights the large number of still uncharacterized viruses in mammals., Author Summary Rodents are the natural reservoir of numerous zoonotic viruses causing serious diseases in humans. We used an unbiased metagenomic approach to characterize the viral diversity in rodent feces. In addition to diet-derived insect and plant viruses mammalian viral sequences were abundant and diverse. Most notably, multiple new circular viral DNA families, two new picornaviridae genera, and the first murine astrovirus and picobirnaviruses were characterized. A mouse kobuvirus was a close relative to the Aichi virus human pathogen. This study significantly increases the known genetic diversity of eukaryotic viruses in rodents and provides an initial description of their enteric viromes.