Explore the vast range of titles available.

Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F . tularensis , the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F . novicida . Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F . novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F . novicida .

subsp. is an intracellular bacterial pathogen and the causative agent of the life-threatening zoonotic disease tularemia. The Pathogenicity Island encodes a large secretion apparatus, known as a Type VI Secretion System (T6SS), which is essential for to escape from its phagosome and multiply within host macrophages and to cause disease in animals. The T6SS, found in one-quarter of Gram-negative bacteria including many highly pathogenic ones, is a recently discovered secretion system that is not yet fully understood. Nevertheless, there have been remarkable advances in our understanding of the structure, composition, and function of T6SSs of several bacteria in the past few years. The system operates like an inside-out headless contractile phage that is anchored to the bacterial membrane via a baseplate and membrane complex. The system injects effector molecules across the inner and outer bacterial membrane and into host prokaryotic or eukaryotic targets to kill, intoxicate, or in the case of , hijack the target cell. Recent advances include an atomic model of the contractile sheath, insights into the mechanics of sheath contraction, the composition of the baseplate and membrane complex, the process of assembly of the apparatus, and identification of numerous effector molecules and activities. While T6SS appears to be an outlier among T6SSs, with limited or no sequence homology with other systems, its structure and organization are strikingly similar to other systems. Nevertheless, we have only scratched the surface in uncovering the mysteries of the T6SS, and there are numerous questions that remain to be answered.

is a highly infectious Gram-negative bacterium that causes tularemia in humans and animals. It has a remarkable ability to survive and replicate within a wide range of host cells. shares many characteristics with of . However, it is rarely pathogenic in humans, and its reduced virulence makes it a suitable model organism for studying infection. This study aimed to identify the pathogenic factors of . Using a novel infection model with HeLa cells expressing FcγRII (HeLa-FcγRII cells), we screened 2,232 transposon mutants of pre-treated with antiserum containing antibodies to find less cytotoxicity strains. The transposon insertion site was identified by sequencing, leading to the determination of the genes responsible for the attenuated cytotoxicity. Additionally, the intracellular behavior of the mutant was investigated within both HeLa-FcγRII and THP-1 cells. A total of thirteen mutants with attenuated cytotoxicity were isolated, and their responsible genes were identified. They are , , and one unnamed gene (FTN_0096). We focused on the gene. The wild-type (WT) strain showed intracellular replication in HeLa-FcγRII and THP-1 cells, but the number of intracellular mutants decreased. The mutant could not escape from phagolysosomes in the initial phases of infection and was digested within the lysosome. The mutant was also detected in the mitochondria and the Golgi complex. The cytokine response induced by mutant was comparable to that of the WT strain. These findings indicate that is important for the intracellular replication of .

Francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. In view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. In this study a shared probe microarray was designed that could be used to compare the transcriptomes of Francisella tularensis subsp. tularensis Schu S4 (Ftt), Francisella tularensis subsp. holarctica OR960246 (Fth), Francisella tularensis subsp. holarctica LVS (LVS), and Francisella novicida U112 (Fn). To gain insight into expression differences that may be related to the differences in virulence of these subspecies, transcriptomes were measured from each strain grown in vitro under identical conditions, utilizing a shared probe microarray. The human avirulent Fn strain exhibited high levels of transcription of genes involved in general metabolism, which are pseudogenes in the human virulent Ftt and Fth strains, consistent with the process of genome decay in the virulent strains. Genes encoding an efflux system (emrA2 cluster of genes), siderophore (fsl operon), acid phosphatase, LPS synthesis, polyamine synthesis, and citrulline ureidase were all highly expressed in Ftt when compared to Fn, suggesting that some of these may contribute to the relative high virulence of Ftt. Genes expressed at a higher level in Ftt when compared to the relatively less virulent Fth included genes encoding isochorismatases, cholylglycine hydrolase, polyamine synthesis, citrulline ureidase, Type IV pilus subunit, and the Francisella Pathogenicity Island protein PdpD. Fth and LVS had very few expression differences, consistent with the derivation of LVS from Fth. This study demonstrated that a shared probe microarray designed to detect transcripts in multiple species/subspecies of Francisella enabled comparative transcriptional analyses that may highlight critical differences that underlie the relative pathogenesis of these strains for humans. This strategy could be extended to other closely-related bacterial species for inter-strain and inter-species analyses.

, a highly virulent bacteria that causes the zoonotic disease tularemia, is considered a potential agent of biological warfare and bioterrorism. Although the host range for several species within the is known, little is known about the natural reservoirs of various species. The lack of knowledge regarding the environmental fates of these pathogens greatly reduces the possibilities for microbial risk assessments. The greater wax moth ( is an insect of the order that has been used as an alternative model to study microbial infection during recent years. The aim of this study was to evaluate as a model system for studies of human pathogenic and closely related opportunistic and non-pathogenic strains within the genus. The employed larvae model demonstrated differences in lethality between human pathogenic and human non-pathogenic or opportunistic species. The and strains were significantly more virulent in the model than the strains of human pathogens and . Our data show that is a possible of insect immunity for studies of both opportunistic and virulent lineages of spp., that produces inverse results regarding lethality in and incapacitating disease in humans. The results provide insight into the potential host specificity of and closely related members of the same genus, thus increasing our present understanding of spp. ecology.

The highly infectious bacteria, Francisella tularensis, colonize a variety of organs and replicate within both phagocytic as well as non-phagocytic cells, to cause the disease tularemia. These microbes contain a conserved cluster of important virulence genes referred to as the Francisella Pathogenicity Island (FPI). Two of the most characterized FPI genes, iglC and pdpA, play a central role in bacterial survival and proliferation within phagocytes, but do not influence bacterial internalization. Yet, their involvement in non-phagocytic epithelial cell infections remains unexplored. To examine the functions of IglC and PdpA on bacterial invasion and replication during epithelial cell infections, we infected liver and lung epithelial cells with F. novicida and F. tularensis 'Type B' Live Vaccine Strain (LVS) deletion mutants (ΔiglC and ΔpdpA) as well as their respective gene complements. We found that deletion of either gene significantly reduced their ability to invade and replicate in epithelial cells. Gene complementation of iglC and pdpA partially rescued bacterial invasion and intracellular growth. Additionally, substantial LAMP1-association with both deletion mutants was observed up to 12 h suggesting that the absence of IglC and PdpA caused deficiencies in their ability to dissociate from LAMP1-positive Francisella Containing Vacuoles (FCVs). This work provides the first evidence that IglC and PdpA are important pathogenic factors for invasion and intracellular growth of Francisella in epithelial cells, and further highlights the discrete mechanisms involved in Francisella infections between phagocytic and non-phagocytic cells.

is an intracellular pathogen for many animals causing the infectious disease, tularemia. Whereas subsp. is highly pathogenic for humans, is almost avirulent for humans, but virulent for mice. In order to compare metabolic fluxes between these strains, we performed C-labeling experiments with subsp. wild type (beaver isolate), subsp. strain LVS, or strain U112 in complex media containing either [U- C ]glucose, [1,2- C ]glucose, [U- C ]serine, or [U- C ]glycerol. GC/MS-based isotopolog profiling of amino acids, polysaccharide-derived glucose, free fructose, amino sugars derived from the cell wall, fatty acids, 3-hydroxybutyrate, lactate, succinate and malate revealed uptake and metabolic usage of all tracers under the experimental conditions with glucose being the major carbon source for all strains under study. The labeling patterns of the subsp. wild type were highly similar to those of the LVS strain, but showed remarkable differences to the labeling profiles of the metabolites from the strain. Glucose was directly used for polysaccharide and cell wall biosynthesis with higher rates in subsp. or metabolized, with higher rates in glycolysis and the non-oxidative pentose phosphate pathway (PPP). Catabolic turnover of glucose gluconeogenesis was also observed. In all strains, Ala was mainly synthesized from pyruvate, although no pathway from pyruvate to Ala is annotated in the genomes of and . Glycerol efficiently served as a gluconeogenetic substrate in , but only less in the subsp. strains. In any of the studied strains, serine did not serve as a major substrate and was not significantly used for gluconeogenesis under the experimental conditions. Rather, it was only utilized, at low rates, in downstream metabolic processes, e.g., acetyl-CoA in the citrate cycle and for fatty acid biosynthesis, especially in the subsp. strains. In summary, the data reflect differential metabolite fluxes in subsp. and suggesting that the different utilization of substrates could be related to host specificity and virulence of .

Ticks are of medical importance owing to their ability to transmit pathogens to humans and animals. The Rocky Mountain wood tick, Dermacentor andersoni , is a vector of a number of pathogens, including Anaplasma marginale, which is the most widespread tick-borne pathogen of livestock. Although ticks host pathogenic bacteria, they also harbor bacterial endosymbionts that have a role in tick physiology, survival, as well as pathogen acquisition and transmission. The goal of this study was to characterize the bacterial microbiome and examine the impact of microbiome disruption on pathogen susceptibility. The bacterial microbiome of two populations of D. andersoni with historically different susceptibilities to A. marginale was characterized. In this study, the microbiome was disrupted and then ticks were exposed to A. marginale or Francisella novicida to determine whether the microbiome correlated with pathogen susceptibility. Our study showed that an increase in proportion and quantity of Rickettsia bellii in the microbiome was negatively correlated to A. marginale levels in ticks. Furthermore, a decrease in Francisella endosymbionts was associated with lower F. novicida infection levels, demonstrating a positive pathogen–endosymbiont relationship. We demonstrate that endosymbionts and pathogens have varying interactions, and suggest that microbiome manipulation may provide a possible method for biocontrol by decreasing pathogen susceptibility of ticks.

Francisella tularensis is the causative agent of the zoonotic disease tularemia. We investigated a pathogenic factor of F . tularensis subsp. novicida (F. novicida). Accordingly, we established a novel infection model using HeLa cells. F. novicida usually infects macrophage lineage cells and less frequently epithelial cells. We successfully infected HeLa cells expressing the Fc receptor (HeLa–FcγRII cells) using F. novicida supplemented with mouse serum containing F. novicida antibodies. A total of 2,232 transposon mutants of F . novicida were screened to determine the relatively fewer cytotoxic strains of the HeLa–FcγRII cells, and 13 strains were thus isolated. Sequencing analysis of transposon insertion sites identified 13 genes, including FTN_0096 . We focused on FTN_0096 . Although the F. novicida wild-type strain proliferated in HeLa–FcγRII and THP-1 cells, the number of intracellular FTN_0096 mutant decreased. FTN_0096 mutant cannot escape from phagolysosomes in the initial phases of infection. Moreover, FTN_0096 mutant was detected in the mitochondria and Golgi complex. These findings indicate the importance of FTN_0096 of F. novicida for intracellular replication in the cells.

Francisella tularensis is an intracellular bacterial pathogen that causes the zoonotic disease tularemia. Following uptake by host cells, the bacteria rapidly escape the phagosome and replicate intracellularly. In previous studies, we found that Francisella produces tubular extensions of its cell surface in response to specific cues and during macrophage infection. In the present study, we used cryogenic electron tomography to examine tube formation by the model Francisella sp., F. novicida . This analysis revealed that tube formation involves extensive bacterial envelope alterations and a dynamic cytoplasmic organelle. Furthermore, tubes produced by bacteria within infected macrophages were associated with the breakdown of the phagosomal membrane. In addition, we found that the Francisella type VI secretion system, which is essential for phagosomal escape, co-localized with the bacterial tubes. These findings reveal the cellular transformations that occur during membrane tubulation by Francisella and suggest a role for the tubes in phagosomal escape.