The Sch9 Kinase Regulates Conidium Size,Stress Responses,and Pathogenesis in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley worldwide. In a previous study on functional characterization of the F. graminearum kinome, one protein kinase gene important for virulence is orthologous to SCH9 that is functionally related to the cAMP-PKA and TOR pathways in the budding yeast. In this study, we further characterized the functions of FgSCH9 in F. graminearum and its ortholog in Magnaporthe oryzae. The ΔFgsch9 mutant was slightly reduced in growth rate but significantly reduced in conidiation, DON production, and virulence on wheat heads and corn silks. It had increased tolerance to elevated temperatures but became hypersensitive to oxidative, hyperosmotic, cell wall, and membrane stresses. The ΔFgsch9 deletion also had conidium morphology defects and produced smaller conidia. These results suggest that FgSCH9 is important for stress responses, DON production, conidiogenesis, and pathogenesis in F. graminearum. In the rice blast fungus Magnaporthe oryzae, the ΔMosch9 mutant also was defective in conidiogenesis and pathogenesis. Interestingly, it also produced smaller conidia and appressoria. Taken together, our data indicate that the SCH9 kinase gene may have a conserved role in regulating conidium size and plant infection in phytopathogenic ascomycetes.     

Identification of a Novel G2073A Mutation in 23S rRNA in Amphenicol-Selected Mutants of Campylobacter jejuni

Objectives

This study was conducted to examine the development and molecular mechanisms of amphenicol resistance in Campylobacter jejuni by using in vitro selection with chloramphenicol and florfenicol. The impact of the resistance development on growth rates was also determined using in vitro culture.

Methods

Chloramphenicol and florfenicol were used as selection agents to perform in vitro stepwise selection. Mutants resistant to the selective agents were obtained from the selection process. The mutant strains were compared with the parent strain for changes in MICs and growth rates. The 23S rRNA gene and the L4 and L22 ribosomal protein genes in the mutant strains and the parent strain were amplified and sequenced to identify potential resistance-associated mutations.

Results

C. jejuni strains that were highly resistant to chloramphenicol and florfenicol were obtained from in vitro selection. A novel G2073A mutation in all three copies of the 23S rRNA gene was identified in all the resistant mutants examined, which showed resistance to both chloramphenicol and florfenicol. In addition, all the mutants selected by chloramphenicol also exhibited the G74D modification in ribosomal protein L4, which was previously shown to confer a low-level erythromycin resistance in Campylobacter species. The mutants selected by florfenicol did not have the G74D mutation in L4. Notably, the amphenicol-resistant mutants also exhibited reduced susceptibility to erythromycin, suggesting that the selection resulted in cross resistance to macrolides.

Conclusions

This study identifies a novel point mutation (G2073A) in 23S rRNA in amphenicol-selected mutants of C. jejuni. Development of amphenicol resistance in Campylobacter likely incurs a fitness cost as the mutant strains showed slower growth rates in antibiotic-free media.     

Flagella-Mediated Adhesion and Extracellular DNA Release Contribute to Biofilm Formation and Stress Tolerance of Campylobacter jejuni

Campylobacter jejuni is a leading cause of foodbourne gastroenteritis, despite fragile behaviour under standard laboratory conditions. In the environment, C. jejuni may survive within biofilms, which can impart resident bacteria with enhanced stress tolerance compared to their planktonic counterparts. While C. jejuni forms biofilms in vitro and in the wild, it had not been confirmed that this lifestyle confers stress tolerance. Moreover, little is understood about molecular mechanisms of biofilm formation in this pathogen. We previously found that a ΔcprS mutant, which carries a deletion in the sensor kinase of the CprRS two-component system, forms enhanced biofilms. Biofilms were also enhanced by the bile salt deoxycholate and contained extracellular DNA. Through more in-depth analysis of ΔcprS and WT under conditions that promote or inhibit biofilms, we sought to further define this lifestyle for C. jejuni. Epistasis experiments with ΔcprS and flagellar mutations (ΔflhA, ΔpflA) suggested that initiation is mediated by flagellum-mediated adherence, a process which was kinetically enhanced by motility. Lysis was also observed, especially under biofilm-enhancing conditions. Microscopy suggested adherence was followed by release of eDNA, which was required for biofilm maturation. Importantly, inhibiting biofilm formation by removal of eDNA with DNase decreased stress tolerance. This work suggests the biofilm lifestyle provides C. jejuni with resilience that has not been apparent from observation of planktonic bacteria during routine laboratory culture, and provides a framework for subsequent molecular studies of C. jejuni biofilms.     

Peripheral CD4+ T Cell Cytokine Responses Following Human Challenge and Re-Challenge with Campylobacter jejuni

Campylobacter jejuni is a leading cause of human gastroenteritis worldwide; however, our understanding of the human immune response to C. jejuni infection is limited. A previous human challenge model has shown that C. jejuni elicits IFNγ production by peripheral blood mononuclear cells, a response associated with protection from clinical disease following re-infection. In this study, we investigate T lymphocyte profiles associated with campylobacteriosis using specimens from a new human challenge model in which C. jejuni-naïve subjects were challenged and re-challenged with C. jejuni CG8421. Multiparameter flow cytometry was used to investigate T lymphocytes as a source of cytokines, including IFNγ, and to identify cytokine patterns associated with either campylobacteriosis or protection from disease. Unexpectedly, all but one subject evaluated re-experienced campylobacteriosis after re-challenge. We show that CD4⁺ T cells make IFNγ and other pro-inflammatory cytokines in response to infection; however, multifunctional cytokine response patterns were not found. Cytokine production from peripheral CD4⁺ T cells was not enhanced following re-challenge, which may suggest deletion or tolerance. Evaluation of alternative paradigms or models is needed to better understand the immune components of protection from campylobacteriosis.     

The Contribution of ArsB to Arsenic Resistance in Campylobacter jejuni

Arsenic, a toxic metalloid, exists in the natural environment and its organic form is approved for use as a feed additive for animal production. As a major foodborne pathogen of animal origin, Campylobacter is exposed to arsenic selection pressure in the food animal production environments. Previous studies showed that Campylobacter isolates from poultry were highly resistant to arsenic compounds and a 4-gene operon (containing arsP, arsR, arsC, and acr3) was associated with arsenic resistance in Campylobacter. However, this 4-gene operon is only present in some Campylobacter isolates and other arsenic resistance mechanisms in C. jejuni have not been characterized. In this study, we determined the role of several putative arsenic resistance genes including arsB, arsC2, and arsR3 in arsenic resistance in C. jejuni and found that arsB, but not the other two genes, contributes to the resistance to arsenite and arsenate. Inactivation of arsB in C. jejuni resulted in 8- and 4-fold reduction in the MICs of arsenite and arsenate, respectively, and complementation of the arsB mutant restored the MIC of arsenite. Additionally, overexpression of arsB in C. jejuni 11168 resulted in a 16-fold increase in the MIC of arsenite. PCR analysis of C. jejuni isolates from different animals hosts indicated that arsB and acr3 (the 4-gene operon) are widely distributed in various C. jejuni strains, suggesting that Campylobacter requires at least one of the two genes for adaptation to arsenic-containing environments. These results identify ArsB as an alternative mechanism for arsenic resistance in C. jejuni and provide new insights into the adaptive mechanisms of Campylobacter in animal food production environments.     

Biological Roles of the O-Methyl Phosphoramidate Capsule Modification in Campylobacter jejuni

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, and the capsular polysaccharide (CPS) of this organism is required for persistence and disease. C. jejuni produces over 47 different capsular structures, including a unique O-methyl phosphoramidate (MeOPN) modification present on most C. jejuni isolates. Although the MeOPN structure is rare in nature it has structural similarity to some synthetic pesticides. In this study, we have demonstrated, by whole genome comparisons and high resolution magic angle spinning NMR, that MeOPN modifications are common to several Campylobacter species. Using MeOPN biosynthesis and transferase mutants generated in C. jejuni strain 81–176, we observed that loss of MeOPN from the cell surface correlated with increased invasion of Caco-2 epithelial cells and reduced resistance to killing by human serum. In C. jejuni, the observed serum mediated killing was determined to result primarily from activation of the classical complement pathway. The C. jejuni MeOPN transferase mutant showed similar levels of colonization relative to the wild-type in chickens, but showed a five-fold drop in colonization when co-infected with the wild-type in piglets. In Galleria mellonella waxmoth larvae, the MeOPN transferase mutant was able to kill the insects at wild-type levels. Furthermore, injection of the larvae with MeOPN-linked monosaccharides or CPS purified from the wild-type strain did not result in larval killing, indicating that MeOPN does not have inherent insecticidal activity.     

Lactobacillus gasseri SBT2055 Reduces Infection by and Colonization of Campylobacter jejuni

Campylobacter is a normal inhabitant of the chicken gut. Pathogenic infection with this organism in humans is accompanied by severe inflammation of the intestinal mucosal surface. The aim of this study was to evaluate the ability of Lactobacillus gasseri SBT2055 (LG2055) to inhibit the adhesion and invasion of Campylobacter jejuni in vitro and to suppress C. jejuni colonization of chicks in vivo. Pretreatment with LG2055 significantly reduced adhesion to and invasion of a human epithelial cell line, Intestine 407, by C. jejuni 81–176. Methanol (MeOH)-fixed LG2055 also reduced infection by C. jejuni 81–176. However, proteinase K (ProK)-treated LG2055 eliminated the inhibitory effects. Moreover, LG2055 co-aggregated with C. jejuni 81–176. ProK treatment prevented this co-aggregation, indicating that the co-aggregation phenotype mediated by the proteinaceous cell-surface components of LG2055 is important for reducing C. jejuni 81–176 adhesion and invasion. In an in vivo assay, oral doses of LG2055 were administered to chicks daily for 14 days after oral inoculation with C. jejuni 81–176. At 14 days post-inoculation, chicks treated with LG2055 had significantly reduced cecum colonization by C. jejuni. Reduction in the number of C. jejuni 81–176 cells adhering to and internalized by human epithelial cells demonstrated that LG2055 is an organism that effectively and competitively excludes C. jejuni 81–176. In addition, the results of the chick colonization assay suggest that treatment with LG2055 could be useful in suppressing C. jejuni colonization of the chicks at early growth stages.     

Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni

Campylobacter jejuni genetic manipulation is restricted by the limited number of antibiotic resistance cassettes available for use in this diarrheal pathogen. In this study, two antibiotic resistance cassettes were developed, encoding for hygromycin B and apramycin resistance, for use in mutagenesis or for selection of gene expression and complementation constructs in C. jejuni. First, the marker genes were successfully modified to allow for insertional mutagenesis or deletion of a gene-of-interest, and were bracketed with restriction sites for the facilitation of site-specific cloning. These hygromycin B and apramycin markers are encoded by plasmids pAC1H and pAC1A, respectively. We also modified an insertional gene-delivery vector to create pRRH and pRRA, containing the hygromycin B and apramycin resistance genes, and 3 unique restriction sites for the directional introduction of genes into the conserved multi-copy rRNA gene clusters of the C. jejuni chromosome. We determined the effective antibiotic concentrations required for selection, and established that no harmful effects or fitness costs were associated with carrying hygromycin B or apramycin resistance under standard C. jejuni laboratory conditions. Using these markers, the arylsulfatase reporter gene astA was deleted, and the ability to genetically complement the astA deletion using pRRH and pRRA for astA gene insertion was demonstrated. Furthermore, the relative levels of expression from the endogenous astA promoter were compared to that of polycistronic mRNA expression from the constitutive promoter upstream of the resistance gene. The development of additional antibiotic resistance cassettes for use in Campylobacter will enable multiple gene deletion and expression combinations as well as more in-depth study of multi-gene systems important for the survival and pathogenesis of this important bacterium.     

Effects of Lipooligosaccharide Inner Core Truncation on Bile Resistance and Chick Colonization by Campylobacter jejuni

Campylobacter jejuni is the most common bacterium that causes diarrhea worldwide, and chickens are considered the main reservoir of this pathogen. This study investigated the effects of serial truncation of lipooligosaccharide (LOS), a major component of the outer membrane of C. jejuni, on its bile resistance and intestinal colonization ability in chickens. Genes encoding manno-heptose synthetases or glycosyltransferases were inactivated to generate isogenic mutants. Serial truncation of the LOS core oligosaccharide caused a stepwise increase in susceptibilities of two C. jejuni strains, NCTC 11168 and 81-176, to bile acids. Inactivation of hldE, hldD, or waaC caused severe truncation of the core oligosaccharide, which greatly increased the susceptibility to bile acids. Both wild-type strains grew normally in chicken intestinal extracts, whereas the mutants with severe oligosaccharide truncation were not detected 12 h after inoculation. These mutants attained viable bacterial counts in the bile acid-free extracts 24 h after inoculation. The wild-type strain 11-164 was present in the cecal contents at >10⁷ CFU/g on 5 days after challenge infection and after this time period, whereas its hldD mutant was present at <10³ CFU/g throughout the experimental period. Trans-complementation of the hldD mutant with the wild-type hldD allele completely restored the in vivo colonization level to that of the wild-type strain. Mutants with a shorter LOS had higher hydrophobicities. Thus, the length of the LOS core oligosaccharide affected the surface hydrophobicity and bile resistance of C. jejuni as well as its ability to colonize chicken intestines.     

Prevention of Biofilm Formation and Removal of Existing Biofilms by Extracellular DNases of Campylobacter jejuni

The fastidious nature of the foodborne bacterial pathogen Campylobacter jejuni contrasts with its ability to survive in the food chain. The formation of biofilms, or the integration into existing biofilms by C. jejuni, is thought to contribute to food chain survival. As extracellular DNA (eDNA) has previously been proposed to play a role in C. jejuni biofilms, we have investigated the role of extracellular DNases (eDNases) produced by C. jejuni in biofilm formation. A search of 2791 C. jejuni genomes highlighted that almost half of C. jejuni genomes contains at least one eDNase gene, but only a minority of isolates contains two or three of these eDNase genes, such as C. jejuni strain RM1221 which contains the cje0256, cje0566 and cje1441 eDNase genes. Strain RM1221 did not form biofilms, whereas the eDNase-negative strains NCTC 11168 and 81116 did. Incubation of pre-formed biofilms of NCTC 11168 with live C. jejuni RM1221 or with spent medium from a RM1221 culture resulted in removal of the biofilm. Inactivation of the cje1441 eDNase gene in strain RM1221 restored biofilm formation, and made the mutant unable to degrade biofilms of strain NCTC 11168. Finally, C. jejuni strain RM1221 was able to degrade genomic DNA from C. jejuni NCTC 11168, 81116 and RM1221, whereas strain NCTC 11168 and the RM1221 cje1441 mutant were unable to do so. This was mirrored by an absence of eDNA in overnight cultures of C. jejuni RM1221. This suggests that the activity of eDNases in C. jejuni affects biofilm formation and is not conducive to a biofilm lifestyle. These eDNases do however have a potential role in controlling biofilm formation by C. jejuni strains in food chain relevant environments.     

Comparison of Predicted Epimerases and Reductases of the Campylobacter jejuni d-altro- and l-gluco-Heptose Synthesis Pathways

Uniquely modified heptoses found in surface carbohydrates of bacterial pathogens are potential therapeutic targets against such pathogens. Our recent biochemical characterization of the GDP-6-deoxy-d-manno- and GDP-6-deoxy-d-altro-heptose biosynthesis pathways has provided the foundation for elucidation of the more complex l-gluco-heptose synthesis pathway of Campylobacter jejuni strain NCTC 11168. In this work we use GDP-4-keto,6-deoxy-d-lyxo-heptose as a surrogate substrate to characterize three enzymes predicted to be involved in this pathway: WcaG_NCTC (also known as Cj1427), MlghB (Cj1430), and MlghC (Cj1428). We compare them with homologues involved in d-altro-heptose production: WcaG₈₁₁₇₆ (formerly WcaG), DdahB (Cjj1430), and DdahC (Cjj1427). We show that despite high levels of similarity, the enzymes have pathway-specific catalytic activities and substrate specificities. MlghB forms three products via C3 and C5 epimerization activities, whereas its DdahB homologue only had C3 epimerase activity along its cognate pathway. MlghC is specific for the double C3/C5 epimer generated by MlghB and produces l-gluco-heptose via stereospecific C4 reductase activity. In contrast, its homologue DdahC only uses the C3 epimer to yield d-altro-heptose via C4 reduction. Finally, we show that WcaG_NCTC is not necessary for l-gluco-heptose synthesis and does not affect its production by MlghB and MlghC, in contrast to its homologue WcaG₈₁₁₇₆, that has regulatory activity on d-altro-heptose synthesis. These studies expand our fundamental understanding of heptose modification, provide new glycobiology tools to synthesize novel heptose derivatives with biomedical applications, and provide a foundation for the structure function analysis of these enzymes.     

Characterisation of a Multi-ligand Binding Chemoreceptor CcmL (Tlp3) of Campylobacter jejuni

Campylobacter jejuni is the leading cause of human gastroenteritis worldwide with over 500 million cases annually. Chemotaxis and motility have been identified as important virulence factors associated with C. jejuni colonisation. Group A transducer-like proteins (Tlps) are responsible for sensing the external environment for bacterial movement to or away from a chemical gradient or stimulus. In this study, we have demonstrated Cj1564 (Tlp3) to be a multi-ligand binding chemoreceptor and report direct evidence supporting the involvement of Cj1564 (Tlp3) in the chemotaxis signalling pathway via small molecule arrays, surface plasmon and nuclear magnetic resonance (SPR and NMR) as well as chemotaxis assays of wild type and isogenic mutant strains. A modified nutrient depleted chemotaxis assay was further used to determine positive or negative chemotaxis with specific ligands. Here we demonstrate the ability of Cj1564 to interact with the chemoattractants isoleucine, purine, malic acid and fumaric acid and chemorepellents lysine, glucosamine, succinic acid, arginine and thiamine. An isogenic mutant of cj1564 was shown to have altered phenotypic characteristics of C. jejuni, including loss of curvature in bacterial cell shape, reduced chemotactic motility and an increase in both autoagglutination and biofilm formation. We demonstrate Cj1564 to have a role in invasion as in in vitro assays the tlp3 isogenic mutant has a reduced ability to adhere and invade a cultured epithelial cell line; interestingly however, colonisation ability of avian caeca appears to be unaltered. Additionally, protein-protein interaction studies revealed signal transduction initiation through the scaffolding proteins CheV and CheW in the chemotaxis sensory pathway. This is the first report characterising Cj1564 as a multi-ligand receptor for C. jejuni, we therefore, propose to name this receptor CcmL, Campylobacter chemoreceptor for multiple ligands. In conclusion, this study identifies a novel multifunctional role for the C. jejuni CcmL chemoreceptor and illustrates its involvement in the chemotaxis pathway and subsequent survival of this organism in the host.     

Role of Alkyl Hydroperoxide Reductase (AhpC) in the Biofilm Formation of Campylobacter jejuni

Biofilm formation of Campylobacter jejuni, a major cause of human gastroenteritis, contributes to the survival of this pathogenic bacterium in different environmental niches; however, molecular mechanisms for its biofilm formation have not been fully understood yet. In this study, the role of oxidative stress resistance in biofilm formation was investigated using mutants defective in catalase (KatA), superoxide dismutase (SodB), and alkyl hydroperoxide reductase (AhpC). Biofilm formation was substantially increased in an ahpC mutant compared to the wild type, and katA and sodB mutants. In contrast to the augmented biofilm formation of the ahpC mutant, a strain overexpressing ahpC exhibited reduced biofilm formation. A perR mutant and a CosR-overexpression strain, both of which upregulate ahpC, also displayed decreased biofilms. However, the introduction of the ahpC mutation to the perR mutant and the CosR-overexpression strain substantially enhanced biofilm formation. The ahpC mutant accumulated more total reactive oxygen species and lipid hydroperoxides than the wild type, and the treatment of the ahpC mutant with antioxidants reduced biofilm formation to the wild-type level. Confocal microscopy analysis showed more microcolonies were developed in the ahpC mutant than the wild type. These results successfully demonstrate that AhpC plays an important role in the biofilm formation of C. jejuni.     

Peptidoglycan-Modifying Enzyme Pgp1 Is Required for Helical Cell Shape and Pathogenicity Traits in Campylobacter jejuni

The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen Campylobacter jejuni displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally. C. jejuni is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated pgp1 (peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for C. jejuni virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of pgp1 resulted in a striking, rod-shaped morphology, making pgp1 the first C. jejuni gene shown to be involved in maintenance of C. jejuni cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped pgp1 mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the pgp1 mutant and a pgp1 overexpressing strain – which similarly produced straight or kinked cells – exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the pgp1 mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a C. jejuni gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics.     

A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics.     

A Novel Protein Kinase Localized to Lipid Droplets Is Required for Droplet Biogenesis in Trypanosomes

Ubiquitous among eukaryotes, lipid droplets are organelles that function to coordinate intracellular lipid homeostasis. Their morphology and abundance is affected by numerous genes, many of which are involved in lipid metabolism. In this report we identify a Trypanosoma brucei protein kinase, LDK, and demonstrate its localization to the periphery of lipid droplets. Association with lipid droplets was abrogated when the hydrophobic domain of LDK was deleted, supporting a model in which the hydrophobic domain is associated with or inserted into the membrane monolayer of the organelle. RNA interference knockdown of LDK modestly affected the growth of mammalian bloodstream-stage parasites but did not affect the growth of insect (procyclic)-stage parasites. However, the abundance of lipid droplets dramatically decreased in both cases. This loss was dominant over treatment with myriocin or growth in delipidated serum, both of which induce lipid body biogenesis. Growth in delipidated serum also increased LDK autophosphorylation activity. Thus, LDK is required for the biogenesis or maintenance of lipid droplets and is one of the few protein kinases specifically and predominantly associated with an intracellular organelle.Trypanosoma brucei is a single-celled eukaryotic pathogen responsible for human African trypanosomiasis (also known as African sleeping sickness) and nagana in domestic animals. More than 50,000 cases of human disease occur yearly, with over 70 million people at risk. No vaccine exists, and chemotherapy is difficult to administer and prone to pathogen resistance. As T. brucei transits between the mammalian bloodstream and the tsetse fly vector during its life cycle, the organism encounters and adapts to profoundly different environmental conditions. The parasite undergoes dramatic changes in both energy (7, 51) and lipid biosynthesis and metabolism (39, 47, 49) as it shifts between these environments.Protein kinases function in numerous regulatory aspects of the cell, including control of the cell cycle and morphology, responses to stress, and transmission of signals from the extracellular environment or between compartments of the cell. As is the case in other eukaryotes, protein kinases, particularly those associated with membranes, are expected to play pivotal roles in the cell''s ability to sense and appropriately respond to its environment. Trypanosoma brucei possesses over 170 protein kinases (16, 44). Most of these can be assigned to the standard groups of protein kinases based on sequence similarity within the kinase domain. However, sequence similarities with kinases from more well-studied organisms are rarely strong enough to allow one-to-one orthologous relationships to be determined (44), and even those which appear orthologous by sequence have sometimes shown functional divergence (46). Hence, an understanding of the roles of specific protein kinases of trypanosomatids requires an individualized assessment. The initial genome analysis of the trypanosomatids (16) showed a lack of receptor tyrosine kinases, but nine T. brucei predicted serine/threonine kinases were annotated as possessing transmembrane domains. One of these was recently shown to be strategically located at a key interface between the host and parasite: the flagellar pocket (38). This eukaryotic translation initiation factor 2α (eIF2α) family kinase was postulated to play a sensory role in monitoring protein transport.Only a very small number of protein kinases of various organisms have been observed to localize to the membranes of intracellular organelles, most of them to the endoplasmic reticulum (ER) (14, 27, 50). Lipid droplets (also known as lipid bodies, adiposomes, or oil bodies in plants) are thought to arise from the ER, although the routes of protein localization to them are not well understood. They are increasingly recognized as legitimate organelles due to their dynamic roles in energy metabolism (40), lipid trafficking (41), and protection against toxic effects of nonesterified lipids and sterols (18). Studies also suggest that they function as potential protein storage depots (12) and in antigen presentation (10). Although recent efforts to expand the lipid droplet proteome have resulted in a vastly increased and in many cases surprising catalogue of potentially associated proteins (3, 5, 11, 12, 23, 37), relatively little is known as to how these structures form and are regulated within the cell.We examine here a novel T. brucei protein kinase with a predicted transmembrane domain. Surprisingly, this protein is localized intracellularly in association with lipid droplets. RNAi-mediated knockdown of this newly identified kinase, dubbed LDK (for lipid droplet kinase), reveals a role in the formation or maintenance of lipid droplets in both mammalian bloodstream-form (BF) and insect procyclic-form (PF) stages of the parasite life cycle.     

Importance of Polyphosphate Kinase 1 for Campylobacter jejuni Viable-but-Nonculturable Cell Formation,Natural Transformation,and Antimicrobial Resistance

Campylobacter jejuni, a gram-negative, microaerophilic bacterium, is a predominant cause of bacterial gastroenteritis in humans. Although considered fragile and fastidious and lacking many classical stress response mechanisms, C. jejuni exhibits a remarkable capacity for survival and adaptation, successfully infecting humans and persisting in the environment. Consequently, understanding the physiological and genetic properties that allow C. jejuni to survive and adapt to various stress conditions is crucial for therapeutic interventions. Of importance is polyphosphate (poly-P) kinase 1 (PPK1), which is a key enzyme mediating the synthesis of poly-P, an essential molecule for survival, mediating stress responses, host colonization, and virulence in many bacteria. Therefore, we investigated the role of PPK1 in C. jejuni pathogenesis, stress survival, and adaptation. Our findings demonstrate that a C. jejuni Δppk1 mutant was deficient in poly-P accumulation, which was associated with a decreased ability to form viable-but-nonculturable cells under acid stress. The Δppk1 mutant also showed a decreased frequency of natural transformation and an increased susceptibility to various antimicrobials. Furthermore, the Δppk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. Complementation of the Δppk1 mutant with the wild-type copy of ppk1 restored the deficient phenotypes to levels similar to those of the wild type. Our results suggest that poly-P plays an important role in stress survival and adaptation and might contribute to genome plasticity and the spread and development of antimicrobial resistance in C. jejuni. These findings highlight the potential of PPK1 as a novel target for therapeutic interventions.Campylobacter jejuni, a gram-negative, microaerophilic bacterium, occurs as a commensal among the intestinal microflora of various animals, especially chickens and cattle (6, 73). However, C. jejuni can infect human hosts, invading the intestinal mucosa and causing watery and/or bloody diarrhea (9). C. jejuni is transmitted to humans primarily through the consumption of contaminated chicken products, raw milk, or water (2, 3). Currently, C. jejuni is considered a leading bacterial cause of human food-borne gastroenteritis (3, 61) and has also been associated with a plethora of symptoms, including acute neuromuscular paralysis (Guillain-Barré syndrome) (26). Since an appropriate vaccine for human campylobacteriosis has yet to be introduced, it has been suggested that C. jejuni infections might be alternatively controlled by reducing colonization in food animals (73). Consequently, determining the physiological and genetic properties that allow the survival of C. jejuni and its colonization of animal hosts, pathogenicity, and adaptation to various stresses is of critical importance.The mechanisms underlying C. jejuni adaptation and survival under stresses imposed by its environment and host are not well understood. High variability between different C. jejuni strains and the unavailability of appropriate genetic tools and animal models have contributed to the lack of knowledge regarding its stress tolerance and pathogenicity. However, it is suggested that the capacity of C. jejuni to form viable-but-nonculturable (VBNC) cells under stress (14) and its readiness for natural transformation (68) and acquiring resistance to antibiotics (39) are among the strategies that promote stress adaptation and survival. Although little is known about the genetics underlying these processes, recent advances in C. jejuni genomics show that this bacterium carries several important genes that might play key roles in mediating stress adaptation and survival. Of particular interest are genes encoding polyphosphate (poly-P) kinases, ppk1 (CJJ81176_1361) and ppk2 (CJJ81176_0633), that were predicted to be involved in the metabolism of poly-P (22, 25, 47), an intracellular granule that impacts several physiological properties in many bacterial species, including pathogenicity, host colonization, adaptation to different environments, and survival (28, 31, 46).Poly-P kinase 1 (PPK1) is encoded by ppk1, which mediates the synthesis of all or most of the poly-P in the cell (33), while ppk2 encodes an enzyme (PPK2) that synthesizes GTP from poly-P (27). Both ppk genes have been associated with the metabolism of poly-P, which consists of phosphate residues that are linked by high-energy phosphoanhydride bonds and is widely distributed in bacterial species (60). Previous reports showed that poly-P plays important roles in bacterial survival and stress tolerance, including ATP production (8), entry of DNA through membrane channels (13, 54), capsule composition (67), maintaining nutritional requirements during starvation (34), motility, biofilm formation, and resistance to oxidative, osmotic, heat, acid and alkaline stresses, and stationary-phase survival (28, 31, 46, 48, 50, 52, 65). Because of their importance in many bacterial species, it is not surprising to assume a role for PPK and poly-P in C. jejuni survival, colonization, and stress tolerance (8).Interestingly, PPK1 has been shown to be important for C. jejuni stress responses and pathogenicity (10). However, the role of ppk1 in key metabolic and physiological responses of C. jejuni still needs further analysis. For instance, it has been proposed that during starvation, poly-P might act as a reservoir for phosphorus and energy (7). Subsequently, poly-P would be crucial for maintaining viability/metabolism in stressed cells. This has been observed in H. pylori, where the occurrence of poly-P correlated with culturability and structurally intact cells (45). Poly-P-containing nonculturable H. pylori showed a capacity for ATP and mRNA synthesis after a nutrient stimulus (45). Consequently, poly-P might be an important factor for the formation of VBNC cells by stressed bacteria, including C. jejuni. Furthermore, natural transformation is perhaps one of the most important mechanisms in the adaptation of C. jejuni, and poly-P has been reported to play a role in the entry of DNA through membrane channels (13, 54). It follows that poly-P might be important for natural transformation, adaptation, and acquisition of antibiotic resistance genes in C. jejuni. Poly-P can further impact the survival and adaptation in C. jejuni by modulating antibiotic resistance properties. For example, poly-P interacted with Escherichia coli ribosomes (42), which are known targets of several antibiotics. These observations suggest that ppk1 might be linked to important physiology and functions such as VBNC cell formation, natural transformation, and antimicrobial resistance in C. jejuni. Therefore, in the present study, we determined the contribution of PPK1 to C. jejuni stress responses and adaptation, including the ability to form VBNC cells under acid stress, natural transformation, and antimicrobial resistance. Furthermore, we assessed the impact of ppk1 deletion on in vivo chicken colonization. Our findings highlight the importance of PPK1 in C. jejuni survival, adaptation to different environmental stresses, and in vivo colonization. These findings also indicate the suitability of PPK1 as a potential target for controlling the proliferation of this pathogen.