IHF regulates host colonization factors in the bee gut symbiont Frischella perrara

Gut bacteria colonize specific niches in the digestive tract of animals. Yet, the genetic basis of these associations often remains elusive. The gut symbiont Frischella perrara colonizes the anterior hindgut of honey bees, where it causes a characteristic immune response leading to the formation of the scab phenotype. Genetic determinants required for the establishment of this specific association are currently unknown. Here, we independently isolated three point mutations in the two genes encoding the DNA-binding protein integration host factor (IHF). The mutations resulted in the formation of larger colonies on agar plates and the absence of an aryl polyene metabolite conferring the yellow color to colonies of F. perrara. Inoculation of microbiota-free bees with one of these mutants drastically decreased gut colonization of F. perrara and abolished scab development. Using RNAseq we show that IHF affects the expression of potential colonization factors, including a colibactin biosynthetic gene cluster, two Type 6 secretion systems, pili genes, and the aryl polyene biosynthesis pathway. Individual gene deletions of these components revealed different colonization defects indicating that these genetic determinants of F. perrara have distinct roles in the interaction with the host. IHF is conserved across many bacteria and may regulate host colonization also in other animal symbionts.

A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species—Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked β1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

The cnf1 gene is associated to an expanding Escherichia coli ST131 H30Rx/C2 sublineage and confers a competitive advantage for host colonization

Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract acting as a source of urinary tract pathogens. We performed a high-throughput genetic screening of predominantly clinical E. coli isolates from wide geographical origins. This revealed a preferential distribution of the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, in four sequence types encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment and has known enhanced capacities to colonize the gastrointestinal tract (GIT). Statistical modeling uncovered a dominant global expansion of cnf1-positive strains within multidrug-resistant ST131 subclade H30Rx/C2. Despite the absence of phylogeographical signals, cnf1-positive isolates adopted a clonal distribution into clusters on the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. Functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2, established that a cnf1-deleted EC131GY is outcompeted by the wildtype strain in a mouse model of competitive infection of the bladder while both strains behave similarly during monoinfections. This points for positive selection of cnf1 during UTI rather than urovirulence. Wildtype EC131GY also outcompeted the mutant when concurrently inoculated into the gastrointestinal tract, arguing for selection within the gut. Whatever the site of selection, these findings support that the benefit of cnf1 enhancing host colonization by ST131-H30Rx/C2 in turn drives a worldwide dissemination of the cnf1 gene together with extended spectrum of antibiotic resistance genes.

Host Colonization as a Major Evolutionary Force Favoring the Diversity and the Emergence of the Worldwide Multidrug-Resistant Escherichia coli ST131

Until now, there has been no indication that the evolutionary dynamics of Escherichia coli ST131 may reflect independent and host-specific adaptation of this lineage outside humans. In contrast, the limited number of ST131 reports in animals supported the common view that it rather reflects a spillover of the human sector.

Differential regulation and production of secondary metabolites among isolates of the fungal wheat pathogen Zymoseptoria tritici

The genome of the wheat pathogenic fungus, Zymoseptoria tritici, represents extensive presence-absence variation in gene content. Here, we addressed variation in biosynthetic gene clusters (BGCs) content and biochemical profiles among three isolates. We analysed secondary metabolite properties based on genome, transcriptome and metabolome data. The isolates represent highly distinct genome architecture, but harbor similar repertoire of BGCs. Expression profiles for most BGCs show comparable patterns of regulation among the isolates, suggesting a conserved 'biochemical infection program'. For all three isolates, we observed a strong up-regulation of an abscisic acid (ABA) gene cluster during biotrophic host colonization, indicating that Z. tritici potentially interfere with host defenses by the biosynthesis of this phytohormone. Further, during in vitro growth the isolates show similar metabolomes congruent with the predicted BGC content. We assessed if secondary metabolite production is regulated by histone methylation using a mutant impaired in formation of facultative heterochromatin (H3K27me3). In contrast to other ascomycete fungi, chromatin modifications play a less prominent role in regulation of secondary metabolites. In summary, we show that Z. tritici has a conserved program of secondary metabolite production contrasting the immense variation in effector expression, some of these metabolites might play a key role during host colonization.

Hybrid histidine kinase BinK represses Vibrio fischeri biofilm signaling at multiple developmental stages

The symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and its exclusive light-organ symbiont, Vibrio fischeri, provides a natural system in which to study host-microbe specificity and gene regulation during the establishment of a mutually-beneficial symbiosis. Colonization of the host relies on bacterial biofilm-like aggregation in the squid mucus field. Symbiotic biofilm formation is controlled by a two-component signaling (TCS) system consisting of regulators RscS-SypF-SypG, which together direct transcription of the Syp symbiotic polysaccharide. TCS systems are broadly important for bacteria to sense environmental cues and then direct changes in behavior. Previously, we identified hybrid histidine kinase BinK as a strong negative regulator of V. fischeri biofilm regulation, and here we further explore the function of BinK. To inhibit biofilm formation, BinK requires the predicted phosphorylation sites in both the histidine kinase (H362) and receiver (D794) domains. Furthermore, we show that RscS is not essential for host colonization when binK is deleted from strain ES114, and imaging of aggregate size revealed no benefit to the presence of RscS in a background lacking BinK. Strains lacking RscS still suffered in competition. Finally, we show that BinK functions to inhibit biofilm gene expression in the light organ crypts, providing evidence for biofilm gene regulation at later stages of host colonization. Overall, this study provides direct evidence for opposing activities of RscS and BinK and yields novel insights into biofilm regulation during the maturation of a beneficial symbiosis. IMPORTANCE Bacteria are often in a biofilm state, and transitions between planktonic and biofilm lifestyles are important for pathogenic, beneficial, and environmental microbes. The critical nature of biofilm formation during Vibrio fischeri colonization of the Hawaiian bobtail squid light organ provides an opportunity to study development of this process in vivo using a combination of genetic and imaging approaches. The current work refines the signaling circuitry of the biofilm pathway in V. fischeri, provides evidence that biofilm regulatory changes occur in the host, and identifies BinK as one of the regulators of that process. This study provides information about how bacteria regulate biofilm gene expression in an intact animal host.

Hybrid histidine kinase BinK represses Vibrio fischeri biofilm signaling at multiple developmental stages

ABSTRACTThe symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and its exclusive light-organ symbiont, Vibrio fischeri, provides a natural system in which to study host-microbe specificity and gene regulation during the establishment of a mutually-beneficial symbiosis. Colonization of the host relies on bacterial biofilm-like aggregation in the squid mucus field. Symbiotic biofilm formation is controlled by a two-component signaling (TCS) system consisting of regulators RscS-SypF-SypG, which together direct transcription of the Syp symbiotic polysaccharide. TCS systems are broadly important for bacteria to sense environmental cues and then direct changes in behavior. Previously, we identified hybrid histidine kinase BinK as a strong negative regulator of V. fischeri biofilm regulation, and here we further explore the function of BinK. To inhibit biofilm formation, BinK requires the predicted phosphorylation sites in both the histidine kinase (H362) and receiver (D794) domains. Furthermore, we show that a strain lacking BinK yields RscS non-essential for host colonization, and imaging of aggregate size revealed no benefit to the presence of RscS in a background lacking BinK. Strains lacking RscS still suffered in competition, suggesting another function for the protein. Finally, we show that BinK functions to inhibit biofilm gene expression in the light organ crypts, providing evidence for biofilm gene regulation at later stages of host colonization. Overall, this study provides direct evidence for opposing activities of RscS and BinK and yields novel insights into biofilm regulation during the maturation of a beneficial symbiosis.IMPORTANCEBacteria are often in a biofilm state, and transitions between planktonic and biofilm lifestyles are important for pathogenic, beneficial, and environmental microbes. The critical nature of biofilm formation during Vibrio fischeri colonization of the Hawaiian bobtail squid light organ provides an opportunity to study development of this process in vivo using a combination of genetic and imaging approaches. The current work refines the signaling circuitry of the biofilm pathway in V. fischeri, provides evidence that biofilm regulatory changes occur in the host, and identifies BinK as one of the regulators of that process. This study provides information about how bacteria regulate biofilm gene expression in an intact animal host.

Rickettsia-host interaction: strategies of intracytosolic host colonization

ABSTRACT Bacterial infection is a highly complex biological process involving a dynamic interaction between the invading microorganism and the host. Specifically, intracellular pathogens seize control over the host cellular processes including membrane dynamics, actin cytoskeleton, phosphoinositide metabolism, intracellular trafficking and immune defense mechanisms to promote their host colonization. To accomplish such challenging tasks, virulent bacteria deploy unique species-specific secreted effectors to evade and/or subvert cellular defense surveillance mechanisms to establish a replication niche. However, despite superficially similar infection strategies, diverse Rickettsia species utilize different effector repertoires to promote host colonization. This review will discuss our current understandings on how different Rickettsia species deploy their effector arsenal to manipulate host cellular processes to promote their intracytosolic life within the mammalian host.

De novo purine nucleotide biosynthesis mediated by amidophosphoribosyl transferase is required for conidiation and essential for the successful host colonization of Magnaporthe oryzae

Amidophosphoribosyl transferase catalyzes the first step of the purine nucleotide biosynthesis by converting 5-phosphoribosyl-1-pyrophosphate into 5-phosphoribosyl-1-amine. In this study, we identified and characterized the functions of MoAde4, an ortholog of yeast Ade4 in the rice blast fungus. MoAde4 is a 537-amino acid protein containing the GATase_6 and pribosyltran domains. Quantitative real-time PCR analysis showed MoADE4 transcripts were highly expressed during conidiation, early-infection, and late-infection stages of the fungus. Disruption of MoADE4 gene resulted in ΔMoade4 mutant exhibiting adenine, adenosine, and hypoxanthine auxotrophy on MM. Conidia quantification assays showed ΔMoade4 mutant was significantly reduced in sporulation. The conidia of ΔMoade4 mutant could still form appressoria but mostly failed to penetrate the rice cuticle. Pathogenicity test showed ΔMoade4 was completely nonpathogenic on rice and barley leaves which was attributed by failure of its infectious hyphae to colonize the host cells. The ΔMoade4 was defective in induction of strong host immunity and had its purine transporter genes repressed during in planta infection. Addition of exogenous adenine partially rescued conidiation and pathogenicity defects of the ΔMoade4 mutant on the barley and rice leaves. Localization assays showed that MoAde4 is located in the cytoplasm. Taken together, our results demonstrate that purine biosynthesis orchestrated by MoAde4 is required for fungal development, conidiation, more importantly, we found it to be essential for fungal pathogenicity not because of the appressorial formation, but appressorium penetration and host colonization during the plant infection of M. oryzae. Thus this findings suggests that purine biosynthesis could act as an important target for combating recalcitrant plant fungal pathogens.