Change is good: variations in common biological mechanisms in the epsilonproteobacterial genera Campylobacter and Helicobacter

Abstract

Microbial evolution and subsequent species diversification enable bacterial organisms to perform common biological processes by a variety of means. The epsilonproteobacteria are a diverse class of prokaryotes that thrive in diverse habitats. Many of these environmental niches are labeled as extreme, whereas other niches include various sites within human, animal, and insect hosts. Some epsilonproteobacteria, such as Campylobacter jejuni and Helicobacter pylori, are common pathogens of humans that inhabit specific regions of the gastrointestinal tract. As such, the biological processes of pathogenic Campylobacter and Helicobacter spp. are often modeled after those of common enteric pathogens such as Salmonella spp. and Escherichia coli. While many exquisite biological mechanisms involving biochemical processes, genetic regulatory pathways, and pathogenesis of disease have been elucidated from studies of Salmonella spp. and E. coli, these paradigms often do not apply to the same processes in the epsilonproteobacteria. Instead, these bacteria often display extensive variation in common biological mechanisms relative to those of other prototypical bacteria. In this review, five biological processes of commonly studied model bacterial species are compared to those of the epsilonproteobacteria C. jejuni and H. pylori. Distinct differences in the processes of flagellar biosynthesis, DNA uptake and recombination, iron homeostasis, interaction with epithelial cells, and protein glycosylation are highlighted. Collectively, these studies support a broader view of the vast repertoire of biological mechanisms employed by bacteria and suggest that future studies of the epsilonproteobacteria will continue to provide novel and interesting information regarding prokaryotic cellular biology.

FIG. 1.

Comparison of the flagellar structures and regulatory cascades of Salmonella spp. and E. coli and those of C. jejuni and H. pylori. Proteins of the flagellar structures are color coded based on which class of the respective genes are found in the flagellar transcriptional hierarchies, using black (class 1), red (class 2), blue (class 3), and gray (outside the flagellar transcriptional hierarchy). Some genes are not exclusive to one class, but these are not included in this figure for simplicity. fliA (encoding σ²⁸) and flgM are included as class 2 genes in C. jejuni, but these genes are not entirely dependent on σ⁵⁴ for expression. Note that not all proteins required for flagellar biosynthesis are shown for simplicity in comparing structures and the transcriptional regulatory cascades. The most current proposed flagellar regulatory cascade for C. jejuni is shown as a representative for H. pylori, but more detailed analysis of the H. pylori system is required to verify this transcriptional hierarchy.

FIG. 2.

Operonic organization of many class 1 genes and che genes of H. pylori with essential or housekeeping genes. Many of these operons have been confirmed to be cotranscribed, as described by Sharma et al. (534). Flagellar genes are indicated in red. Gene designations are based on the genomic sequence of H. pylori strain 26695.

FIG. 3.

Components of the T4P and T2SS involved in transformation. (Left) Model of the T4P system, based on those previously reported (90, 91). Indicated components are named after the N. gonorrhoeae T4P. The outer membrane-spanning pore complex consists of PilQ and is assisted by the PilP pilot protein. The pilus/pseudopilus structure is composed of major (PilE) and minor (PilV or ComP) pilin proteins, which are processed by the prepilin peptidase, PilD. The T4P adhesin, PilC, associates with the distal end of the pilus structure (not shown). Pilus/pseudopilus biogenesis is also facilitated by the traffic NTPase PilF and the inner membrane protein PilG. Another traffic NTPase, PilT, is involved in disassembly of the pilus/pseudopilus. During twitching motility, the pilus complex polymerizes to facilitate attachment to a nearby surface. Once attached, the pilus complex depolymerizes to result in bacterial locomotion. In a similar manner, donor DNA may be taken into the cell by binding to an unidentified DNA receptor followed by retraction of the competence pseudopilus structure. Once inside the periplasmic space, the donor DNA may then be processed and translocated into the cytoplasm through ComA. DNA translocation may involve the periplasmic protein ComE. (Right) The T2SS contains several components that are homologous to the T4P system. The components shown are referred to by their respective letter designations, and the model is based on those reported previously (91, 516). Note that not all known T2SS components are shown and that some components may be species specific. Assisted by the S pilot protein, protein D forms the outer membrane-spanning pore complex. The pseudopilus-like structure is formed by G, as well as other minor pseudopilins, collectively represented by T, which are processed by the prepilin peptidase O. Other components involved in pseudopilus assembly are the inner membrane protein F and the traffic NTPase, E. The secretion-activating signal may be transmitted from E to the outer membrane by proteins C, M, and L (not shown). Known components of the C. jejuni Cts T2SS-like transformation system are depicted in blue. These components include homologs of the pore protein D (CtsD), the major and minor pseudopilins G (CtsG) and T (CtsT), the inner membrane protein F (CtsF), and the traffic NTPase E (CtsE) (641). Additionally, the periplasmic protein Cj0011c may be involved in transport of DNA in the periplasm. It is proposed that the T2SS pseudopilus acts as a piston, forcing secreted effectors from the periplasm into the extracellular environment. Although the mechanism for T2SS-mediated DNA uptake is currently unknown, it is possible that once donor DNA is bound, the T2SS works in reverse to transport DNA across the outer membrane and into the recipient cell.

FIG. 4.

T4SS-related DNA uptake system of H. pylori and C. jejuni. The model of T4SS-mediated DNA uptake in H. pylori and C. jejuni is based on those proposed previously (261, 263, 548, 573). All components shown have been identified in H. pylori. The components also identified in C. jejuni are shown in blue (31). The transmembrane pore complex is composed of the Com proteins ComB6, ComB7, ComB8, ComB9, and ComB10. ComB3 is thought to be located in the inner membrane, but its function is unclear. Likewise, the precise function of ComB2 is not known; however, it is proposed that ComB2 forms the surface-exposed region of the T4SS. The ComB4 ATPase is thought to exist in a complex with ComB3 and may provide the energy necessary for T4SS biogenesis and/or substrate translocation. Once bound, through an unknown mechanism, donor DNA is taken up through the T4SS transmembrane complex into the periplasm. The periplasmic DNA is then processed and is likely translocated into the cytoplasm by the inner membrane-spanning channel ComEC. The processed single-stranded DNA can then be used as a substrate for homologous recombination.

FIG. 5.

dsDNA processing in homologous recombination. A simplified model of homologous recombination is shown. Component names in black are common to E. coli, H. pylori, and C. jejuni, those in red are found only in H. pylori and C. jejuni, and those in blue are found only in E. coli. (A) dsDNA is recognized and processed by either the RecBCD (in E. coli) or AddAB (in H. pylori and C. jejuni) complex. As the two strands become separated, the 5′ end is degraded preferentially, allowing RecA and SSB to bind to the exposed 3′ tail, forming the presynaptic complex. RecJ may also be involved in the conversion of dsDNA to a ssDNA substrate for RecA processing. (B) RecA then mediates the strand invasion reaction, which forms the joint molecule that scans the recipient DNA until a region of sufficient homology is found. (C) Holliday junction formation and branch migration are catalyzed by RuvAB. In E. coli, RecG also binds the Holliday junction structure and facilitates branch migration; however, H. pylori RecG (and possibly that of C. jejuni) binds the junction and inhibits branch migration and resolution. (D) The migrating DNA heteroduplex is recognized by the RuvC resolvase and is resolved to form either spliced or patched recombinant DNA molecules.

FIG. 6.

Genetic organization and simplified regulation of the E. coli fur locus. (A) The primary fur promoter lies directly upstream of the fur coding region. Transcription typically initiates from one of two proximally located transcriptional start sites (TSSs), as indicated by P_fur1, and is regulated by Fur, Cap, and/or OxyR (131, 667). A secondary SoxR-regulated promoter upstream of the fldA coding sequence may also drive expression of fur under oxidative stress conditions (667). (B) (Top) Under conditions of oxidative stress, fur expression is activated from the proximal promoter TSSs (P_fur1) and/or from the fldA promoter (P_fur2) by SoxR (667), resulting in the production of either a monocistronic mRNA (mRNA1) or a polycistronic mRNA that also contains fldA (mRNA2). (Middle) When excess iron is present, fur expression is repressed in an autoregulatory manner. (Bottom) fur expression from the proximal promoter is also activated in response to increases in cAMP, which results in the production of a monocistronic mRNA.

FIG. 7.

Genetic organization of the C. jejuni fur locus. In the absence of its own promoter, fur expression in C. jejuni is driven by one of two distal promoters. Transcription initiated from the P₁ promoter results in the production of a polycistronic mRNA containing both cj0399 and fur coding regions (mRNA1). Alternatively, expression from the P₂ promoter produces an mRNA carrying gatC, cj0399, and fur (mRNA2). Neither of these promoters is iron responsive or Fur regulated, indicating that fur expression is not autoregulatory but instead responds to unknown environmental signals (612).

FIG. 8.

Autoregulation of H. pylori fur. The model shown was based on the work of Delany et al. (128, 129). (A) The relative positions of the two Fe²⁺-Fur binding sites and the single apo-Fur binding site are indicated. Fe²⁺-Fur binds DNA as a dimer (111, 128, 482); however, the oligomeric state of apo-Fur is unknown, as indicated by the question mark. For simplicity, apo-Fur is shown to function as a monomer. (B) (Top) Under iron-replete conditions, Fe²⁺-Fur binds the high-affinity site in box I and represses fur expression. (Middle) Under conditions of high/excess iron, Fe²⁺-Fur also binds the lower-affinity site in box II, resulting in a more complete repression of fur expression. (Bottom) In the absence of iron or under iron-restricted conditions, box II is not bound by Fur and fur expression is derepressed. In addition, apo-Fur binds the target sequences in box III and box I, activating fur transcription (129).

FIG. 9.

Comparison of the interactions of S. enterica serovar Typhimurium, C. jejuni, EHEC (or EPEC), and H. pylori with epithelial cells. (A) Adherence of Salmonella spp. to epithelial cells is mediated by both fimbrial and nonfimbrial adhesins. Subsequent translocation of effector proteins by the SPI-1 T3SS leads to actin rearrangements that result in membrane ruffling and invasion of the bacterium. Translocation of SPI-2 effectors results in maturation of the initial vesicle to the SCV. The SCV is modified, resulting in the formation of Sifs and migration of the SCV to a location near the Golgi apparatus to serve as an intracellular replicative niche. (B) C. jejuni adherence likely involves flagellar motility and a collection of outer membrane proteins. Subsequent invasion and intracellular survival mechanisms may be mediated by a variety of proteins, with some possibly secreted by the flagellar machinery. Invasion of host cells by C. jejuni is a microtubule-dependent process that results in membrane protrusions. C. jejuni initially enters the host cell in an endocytic vacuole that matures to a specialized CCV. The CCV is trafficked to a perinuclear location in a process involving microtubules. Question marks indicate factors and steps in CCV maturation that are unknown. (C) EHEC and EPEC use a variety of pili and other proteins to mediate a localized adherence pattern to intestinal epithelial cells. Subsequent translocation of T3SS effector proteins causes the polymerization of actin filaments and the formation of A/E lesions. A/E lesions are characterized by loss of microvilli and the formation of pedestal structures that promote tight adherence of the bacteria. (D) H. pylori subsists mainly in the mucous layer covering the gastric epithelium. Adherence of H. pylori is influenced by flagellar motility and a series of outer membrane proteins that use blood group antigens as specific receptors. Translocation of CagA and possibly other effectors by the Cag T4SS intoxicates the host cell to result in disruption of normal signaling pathways, loss of polarity, and dysregulation of cellular trafficking. These processes are thought to possibly redirect nutrients to the apical surface to promote growth of H. pylori. Evidence suggests that a small percentage of H. pylori organisms invade host cells and escape a modified vacuole before returning to the mucous layer. Question marks indicate unknown mechanisms of vacuole escape and release from the epithelial cell, as well as factors that may be redirected to the apical surface to promote H. pylori growth.

FIG. 10.

O-linked protein glycosylation systems of Campylobacter and Helicobacter species. The O-linked protein glycosylation systems result in the production of different types of glycans linked to flagellin proteins. The PseAc pathway begins with UDP-GlcNAc and is relatively conserved in Campylobacter and Helicobacter spp. The PseAm pathway of C. jejuni converts CMP-PseAc to CMP-PseAm for addition of the glycan to flagellin. The LegAm pathway of C. coli begins with an unknown sugar to result in production of Leg5Am7Ac or Leg5AmNMe7Ac for addition to flagellins. The pathways of the O-linked glycosylation systems result in the production of nucleotide-linked glycans (CMP-glycans) for addition to flagellins. Due to the production of two different glycans within C. jejuni and C. coli, flagellin proteins are modified heterogeneously with different sugars. GlcNAc, N-acetylglucosamine; PseAc, pseudaminic acid; PseAm, pseudaminic acid with acetamidino addition; LegAm, legionaminic acid; Leg5Am7Ac, legionaminic acid with acetamidino modification; and Leg5AmNMe7Ac, legionaminic acid with N-methylacetamidoyl modification.

FIG. 11.

N-linked protein glycosylation system of C. jejuni. The N-linked protein glycosylation system of C. jejuni begins with UDP-GlcNAc to result in a heptasaccharide that is linked to a lipid carrier on the cytoplasmic face of the inner membrane (IM). The heptasaccharide is flipped to the periplasm by PglK and is removed from the carrier by PglB to result in fOS or linkage to a protein. Glc, glucose; GlcNAc, N-acetylglucosamine; Bac, bacillosamine; GalNAc, N-acetylgalactosamine.