Viscosity-dependent determinants of Campylobacter jejuni impacting the velocity of flagellar motility.

IMPORTANCE: Bacteria can adapt flagellar motor output in response to the load that the extracellular milieu imparts on the flagellar filament to enable propulsion. Bacteria can adapt flagellar motor output in response to the load that the extracellular milieu imparts on the flagellar filament to enable propulsion through diverse environments. These changes may involve increasing power and torque in high-viscosity environments or reducing power and flagellar rotation upon contact with a surface. C. jejuni swimming velocity in low-viscosity environments is comparable to other bacterial flagellates and increases significantly as external viscosity increases. In this work, we provide evidence that the mechanics of the C. jejuni flagellar motor has evolved to naturally promote high swimming velocity in high-viscosity environments. We found that C. jejuni produces VidA and VidB as auxiliary proteins to specifically affect flagellar motor activity in low viscosity to reduce swimming velocity. Our findings provide some of the first insights into different mechanisms that exist in bacteria to alter the mechanics of a flagellar motor, depending on the viscosity of extracellular environments.

Diversification of Campylobacter jejuni Flagellar C-Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes.

Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species.IMPORTANCE The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Campylobacter jejuni flagellar C ring, the mechanistic core of the motor. Our work provides insight into how usually conserved core components may have diversified by gene duplication, enabling a division of labor of the ancestral protein between the two new proteins, acquisition of new roles in flagellar assembly and motility, and expansion of the function of the flagellum beyond motility, including spatial regulation of cell division and numerical control of flagellar biogenesis in C. jejuni Our results highlight that relatively small changes, such as gene duplications, can have substantial ramifications on the cellular roles of a molecular machine.

A Chaperone for the Stator Units of a Bacterial Flagellum.

The stator units of the flagellum supply power to the flagellar motor via ion transport across the cytoplasmic membrane and generate torque on the rotor for rotation. Flagellar motors across bacterial species have evolved adaptations that impact and enhance stator function to meet the demands of each species, including producing stator units using different fuel types or various stator units for different motility modalities. Campylobacter jejuni produces one of the most complex and powerful flagellar motors by positioning 17 stator units at a greater radial distance than in most other bacteria to increase power and torque for high velocity of motility. We report another evolutionary adaptation impacting flagellar stators by identifying FlgX as a chaperone for C. jejuni stator units to ensure sufficient power and torque for flagellar rotation and motility. We discovered that FlgX maintains MotA and MotB stator protein integrity likely through a direct interaction with MotA that prevents their degradation. Suppressor analysis suggested that the physiology of C. jejuni drives the requirement for FlgX to protect stator units from proteolysis by the FtsH protease complex. C. jejuni ΔflgX was strongly attenuated for colonization of the natural avian host, but colonization capacity was greatly restored by a single mutation in MotA. These findings suggest that the likely sole function of FlgX is to preserve stator unit integrity for the motility required for host interactions. Our findings demonstrate another evolved adaptation in motile bacteria to ensure the equipment of the flagellar motor with sufficient power to generate torque for motility.IMPORTANCE The bacterial flagellum is a reversible rotating motor powered by ion transport through stator units, which also exert torque on the rotor component to turn the flagellum for motility. Species-specific adaptations to flagellar motors impact stator function to meet the demands of each species to sufficiently power flagellar rotation. We identified another evolutionary adaptation by discovering that FlgX of Campylobacter jejuni preserves the integrity of stator units by functioning as a chaperone to protect stator proteins from degradation by the FtsH protease complex due to the physiology of the bacterium. FlgX is required to maintain a level of stator units sufficient to power the naturally high-torque flagellar motor of C. jejuni for motility in intestinal mucosal layers to colonize hosts. Our work continues to identify an increasing number of adaptations to flagellar motors across bacterial species that provide the mechanics necessary for producing an effective rotating nanomachine for motility.

Microbiota-Derived Short-Chain Fatty Acids Modulate Expression of Campylobacter jejuni Determinants Required for Commensalism and Virulence.

Campylobacter jejuni promotes commensalism in the intestinal tracts of avian hosts and diarrheal disease in humans, yet components of intestinal environments recognized as spatial cues specific for different intestinal regions by the bacterium to initiate interactions in either host are mostly unknown. By analyzing a C. jejuni acetogenesis mutant defective in converting acetyl coenzyme A (Ac-CoA) to acetate and commensal colonization of young chicks, we discovered evidence for in vivo microbiota-derived short-chain fatty acids (SCFAs) and organic acids as cues recognized by C. jejuni that modulate expression of determinants required for commensalism. We identified a set of C. jejuni genes encoding catabolic enzymes and transport systems for amino acids required for in vivo growth whose expression was modulated by SCFAs. Transcription of these genes was reduced in the acetogenesis mutant but was restored upon supplementation with physiological concentrations of the SCFAs acetate and butyrate present in the lower intestinal tracts of avian and human hosts. Conversely, the organic acid lactate, which is abundant in the upper intestinal tract where C. jejuni colonizes less efficiently, reduced expression of these genes. We propose that microbiota-generated SCFAs and lactate are cues for C. jejuni to discriminate between different intestinal regions. Spatial gradients of these metabolites likely allow C. jejuni to locate preferred niches in the lower intestinal tract and induce expression of factors required for intestinal growth and commensal colonization. Our findings provide insights into the types of cues C. jejuni monitors in the avian host for commensalism and likely in humans to promote diarrheal disease.IMPORTANCECampylobacter jejuni is a commensal of the intestinal tracts of avian species and other animals and a leading cause of diarrheal disease in humans. The types of cues sensed by C. jejuni to influence responses to promote commensalism or infection are largely lacking. By analyzing a C. jejuni acetogenesis mutant, we discovered a set of genes whose expression is modulated by lactate and short-chain fatty acids produced by the microbiota in the intestinal tract. These genes include those encoding catabolic enzymes and transport systems for amino acids that are required by C. jejuni for in vivo growth and intestinal colonization. We propose that gradients of these microbiota-generated metabolites are cues for spatial discrimination between areas of the intestines so that the bacterium can locate niches in the lower intestinal tract for optimal growth for commensalism in avian species and possibly infection of human hosts leading to diarrheal disease.

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.

Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.

FlhG employs diverse intrinsic domains and influences FlhF GTPase activity to numerically regulate polar flagellar biogenesis in Campylobacter jejuni.

Flagellation in polar flagellates is one of the rare biosynthetic processes known to be numerically regulated in bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuni FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. Unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni. Instead, two regions of C. jejuni FlhG that are absent or significantly altered in FlhG orthologs are involved in numerical regulation of flagellar biogenesis. Additionally, we found that C. jejuni FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. Our work suggests that FlhG ATPases divergently evolved in each polarly flagellated species to employ different intrinsic domains and extrinsic effectors to ultimately mediate a common output - precise numerical control of polar flagellar biogenesis required to create species-specific flagellation patterns optimal for motility.

Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport.

Campylobacter jejuni is a leading cause of diarrheal disease in humans and an intestinal commensal in poultry and other agriculturally important animals. These zoonotic infections result in significant amounts of C. jejuni present in the food supply to contribute to disease in humans. We previously found that a transposon insertion in Cjj81176_1038, encoding a homolog of the Escherichia coli LivJ periplasmic binding protein of the leucine, isoleucine, and valine (LIV) branched-chain amino acid transport system, reduced the commensal colonization capacity of C. jejuni 81-176 in chicks. Cjj81176_1038 is the first gene of a six-gene locus that encodes homologous components of the E. coli LIV system. By analyzing mutants with in-frame deletions of individual genes or pairs of genes, we found that this system constitutes a LIV transport system in C. jejuni responsible for a high level of leucine acquisition and, to a lesser extent, isoleucine and valine acquisition. Despite each LIV protein being required for branched-chain amino acid transport, only the LivJ and LivK periplasmic binding proteins were required for wild-type levels of commensal colonization of chicks. All LIV permease and ATPase components were dispensable for in vivo growth. These results suggest that the biological functions of LivJ and LivK for colonization are more complex than previously hypothesized and extend beyond a role for binding and acquiring branched-chain amino acids during commensalism. In contrast to other studies indicating a requirement and utilization of other specific amino acids for colonization, acquisition of branched-chain amino acids does not appear to be a determinant for C. jejuni during commensalism.

Functional analysis of the RdxA and RdxB nitroreductases of Campylobacter jejuni reveals that mutations in rdxA confer metronidazole resistance.

Campylobacter jejuni is a leading cause of gastroenteritis in humans and a commensal bacterium of the intestinal tracts of many wild and agriculturally significant animals. We identified and characterized a locus, which we annotated as rdxAB, encoding two nitroreductases. RdxA was found to be responsible for sensitivity to metronidazole (Mtz), a common therapeutic agent for another epsilonproteobacterium, Helicobacter pylori. Multiple, independently derived mutations in rdxA but not rdxB resulted in resistance to Mtz (Mtz(r)), suggesting that, unlike the case in H. pylori, Mtz(r) might not be a polygenic trait. Similarly, Mtz(r) C. jejuni was isolated after both in vitro and in vivo growth in the absence of selection that contained frameshift, point, insertion, or deletion mutations within rdxA, possibly revealing genetic variability of this trait in C. jejuni due to spontaneous DNA replication errors occurring during normal growth of the bacterium. Similar to previous findings with H. pylori RdxA, biochemical analysis of C. jejuni RdxA showed strong oxidase activity, with reduction of Mtz occurring only under anaerobic conditions. RdxB showed similar characteristics but at levels lower than those for RdxA. Genetic analysis confirmed that rdxA and rdxB are cotranscribed and induced during in vivo growth in the chick intestinal tract, but an absence of these genes did not strongly impair C. jejuni for commensal colonization. Further studies indicate that rdxA is a convenient locus for complementation of mutants in cis. Our work contributes to the growing knowledge of determinants contributing to susceptibility to Mtz (Mtz(s)) and supports previous observations of the fundamental differences in the activities of nitroreductases from epsilonproteobacteria.

Defining the Mga regulon: Comparative transcriptome analysis reveals both direct and indirect regulation by Mga in the group A streptococcus.

The regulator Mga in the group A streptococcus (GAS) is known to directly activate several virulence genes important for colonization and immune evasion. Transcriptome analysis comparing two mga-1 serotypes (M1 SF370, M6 JRS4) and one mga-2 serotype (M4 GA40634) against their isogenic mga-inactivated strains uncovered a broader Mga regulon profile containing both activated and repressed genes with predicted functions primarily related to sugar metabolism. This was reflected in the altered abilities of M1 and M4 Mga mutants to grow in chemically defined media with a single sugar source compared with their wild-type counterparts. Although the M1 and M4 Mga profiles were similar, the M6 JRS4 was clearly distinct, even from other M6 strains. Real-time RT-PCR and Northern blots confirmed that established core Mga regulon genes directly activated by Mga (emm, scpA, sof, fba) exhibited the highest activation levels across all strains tested. Spy2036 encoding a cytosolic hypothetical protein was highly activated in all three serotypes and was called gene regulated by Mga (grm). Mga bound directly to Pgrm, which overlaps the Mga-regulated Psof in OF+ strains, suggesting that grm is part of the core Mga regulon and Mga is able to activate divergently transcribed genes from a single site. Furthermore, Mga activated speB when detectable in the wild-type strain, although direct binding of Mga to PspeB could not be demonstrated. Thus, Mga is able to both directly and indirectly regulate genes shown to be important for virulence and the metabolic homeostasis of GAS.

Role of Streptococcus pyogenes two-component response regulators in the temporal control of Mga and the Mga-regulated virulence gene emm.

We examined the role of Streptococcus pyogenes two-component response regulators (SptR) in expression of Mga and the Mga-regulated gene emm. Both serotype M6 and serotype M1 mutants in 12 of the 13 identified sptR genes exhibited levels of emm transcripts and Mga protein comparable to those of the wild type during exponential and stationary phases of growth. Thus, temporal control of these virulence genes does not require Spt response regulators.

amrA encodes a putative membrane protein necessary for maximal exponential phase expression of the Mga virulence regulon in Streptococcus pyogenes.

The transcriptional regulator Mga activates a regulon of virulence genes important for colonization and immune evasion in GAS. Using transposon mutagenesis of a serotype M6 group A streptococcus (GAS) reporter strain KSM148, we have identified an open reading frame (ORF) designated amrA that is required for maximal activation of the Mga regulon during exponential phase. A deletion in amrA, but not in the downstream transcriptionally linked ORF Spy0798, was able to reproduce the phenotype seen in the transposon mutants. Northern analysis for mga and emm transcripts, as well as Western analysis of Mga, confirmed a reduction in mga expression leading to a decrease in transcription of the Mga-regulated emm in the amrA deletion and transposon mutants. Furthermore, both the amrA deletion mutant and an original transposon mutant could be complemented using amrA expressed from a nisin-inducible expression system. As amrA is strongly conserved across the sequenced streptococcal M types, and inactivation of amrA in an M3 serotype also resulted in reduction of emm transcripts, the role of amrA does not appear to be serotype specific. Although the specific function of AmrA is unknown, its putative membrane localization and homology to transporters involved in cell wall synthesis suggest a link between growth and virulence gene expression in GAS.

Identification of Aeromonas hydrophila cytotoxic enterotoxin-induced genes in macrophages using microarrays.

A cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses several biological activities, and it induces an inflammatory response in the host. In this study, we used microarrays to gain a global and molecular view of the cellular transcriptional responses to Act and to identify important genes up-regulated by this toxin. Total RNA was isolated at 0, 2, and 12 h from Act-treated macrophages and applied to Affymetrix MGU74 arrays, and the data were processed using a multi-analysis approach to identify genes that might be critical in the inflammatory process evoked by Act. Seventy-six genes were significantly and consistently up-regulated. Many of these genes were immune-related, and several were transcription factors, adhesion molecules, and cytokines. Additionally, we identified several apoptosis-associated genes that were significantly up-regulated in Act-treated macrophages. Act-induced apoptosis of macrophages was confirmed by annexin V staining and DNA laddering. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to verify increased expression of some inflammatory and apoptosis-associated genes identified by the microarray analysis. To further confirm Act-induced increases in gene expression, real-time RT-PCR was also used for selected genes. Taken together, the array data provided for the first time a global view of Act-mediated signal transduction and clearly demonstrated an inflammatory response and apoptosis mediated by this toxin in host cells at the molecular level.

Role of melittin-like region within phospholipase A(2)-activating protein in biological function.

Phospholipase A(2)-activating protein (PLAA) has been implicated in the production of prostaglandins (e.g. PGE(2)) via activation of phospholipases in various stimulated cell types. Human PLAA, with 738 amino acid (aa) residues, contains a region of 38% homology (aa 503-538) with the 26-aa long melittin peptide, a major component of bee venom and a reported regulator of phospholipase A(2) and phospholipase D activity. To learn more about the role of PLAA in the production of eicosanoids and other inflammatory mediators, we synthesized a murine PLAA peptide (36-aa long) having homology to melittin, as well as to human and rat PLAA. The PLAA peptide and melittin increased the expression of genes encoding the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) and cyclooxygenase-2 (COX-2), which is involved in PGE(2) production. We determined that the C-terminal region of the PLAA peptide (aa 515-538) was essential, since truncation of the C-terminal end of the PLAA peptide significantly reduced expression of genes encoding TNFalpha and COX-2 in macrophages. We concluded that PLAA could be important in the regulation of the inflammatory response because of its stimulatory effects on eicosanoid and cytokine synthesis. Consequently, control of plaa gene expression could be a target for the development of new drugs to control the inflammatory response.

Phospholipase A2-activating protein--an important regulatory molecule in modulating cyclooxygenase-2 and tumor necrosis factor production during inflammation.

Inflammation is a complex multifactorial process and a hallmark of many inflammatory diseases. Most of the tissue destruction that occurs in these diseases is the result of an aberrant or often uncontrolled immune response. Factors that play an important role in such diseases include pro-inflammatory cytokines, complement, and eicosanoids. This review focuses on eicosanoids and their regulation via phospholipase A2-activating protein, which could be targeted as a new therapeutic tool to control inflammatory diseases.