Lysinoalanine cross-linking is a conserved post-translational modification in the spirochete flagellar hook.

Spirochetes cause Lyme disease, leptospirosis, syphilis, and several other human illnesses. Unlike other bacteria, spirochete flagella are enclosed within the periplasmic space where the filaments distort and push the cell body by the action of the flagellar motors. We previously demonstrated that the oral pathogen Treponema denticola (Td) and Lyme disease pathogen Borreliella burgdorferi (Bb) form covalent lysinoalanine (Lal) cross-links between conserved cysteine and lysine residues of the FlgE protein that composes the flagellar hook. In Td, Lal is unnecessary for hook assembly but is required for motility, presumably due to the stabilizing effect of the cross-link. Herein, we extend these findings to other, representative spirochete species across the phylum. We confirm the presence of Lal cross-linked peptides in recombinant and in vivo-derived samples from Treponema spp., Borreliella spp., Brachyspira spp., and Leptospira spp. As was observed with Td, a mutant strain of Bb unable to form the cross-link has greatly impaired motility. FlgE from Leptospira spp. does not conserve the Lal-forming cysteine residue which is instead substituted by serine. Nevertheless, Leptospira interrogans FlgE also forms Lal, with several different Lal isoforms being detected between Ser-179 and Lys-145, Lys-148, and Lys-166, thereby highlighting species or order-specific differences within the phylum. Our data reveal that the Lal cross-link is a conserved and necessary posttranslational modification across the spirochete phylum and may thus represent an effective target for the development of spirochete-specific antimicrobials.

Functional and structural analyses reveal that a dual domain sialidase protects bacteria from complement killing through desialylation of complement factors.

The complement system is the first line of innate immune defense against microbial infections. To survive in humans and cause infections, bacterial pathogens have developed sophisticated mechanisms to subvert the complement-mediated bactericidal activity. There are reports that sialidases, also known as neuraminidases, are implicated in bacterial complement resistance; however, its underlying molecular mechanism remains elusive. Several complement proteins (e.g., C1q, C4, and C5) and regulators (e.g., factor H and C4bp) are modified by various sialoglycans (glycans with terminal sialic acids), which are essential for their functions. This report provides both functional and structural evidence that bacterial sialidases can disarm the complement system via desialylating key complement proteins and regulators. The oral bacterium Porphyromonas gingivalis, a "keystone" pathogen of periodontitis, produces a dual domain sialidase (PG0352). Biochemical analyses reveal that PG0352 can desialylate human serum and complement factors and thus protect bacteria from serum killing. Structural analyses show that PG0352 contains a N-terminal carbohydrate-binding module (CBM) and a C-terminal sialidase domain that exhibits a canonical six-bladed ß-propeller sialidase fold with each blade composed of 3-4 antiparallel ß-strands. Follow-up functional studies show that PG0352 forms monomers and is active in a broad range of pH. While PG0352 can remove both N-acetylneuraminic acid (Neu5Ac) and N-glycolyl-neuraminic acid (Neu5Gc), it has a higher affinity to Neu5Ac, the most abundant sialic acid in humans. Structural and functional analyses further demonstrate that the CBM binds to carbohydrates and serum glycoproteins. The results shown in this report provide new insights into understanding the role of sialidases in bacterial virulence and open a new avenue to investigate the molecular mechanisms of bacterial complement resistance.

Anti-σ28 Factor FlgM Regulates Flagellin Gene Expression and Flagellar Polarity of Treponema denticola.

FlgM, an antagonist of FliA (also known as σ28), inhibits transcription of bacterial class 3 flagellar genes. It does so primarily through binding to free σ28 to prevent it from forming a complex with core RNA polymerase. We recently identified an FliA homolog (FliATd) in the oral spirochete Treponema denticola; however, its antagonist FlgM remained uncharacterized. Herein, we provide several lines of evidence that TDE0201 functions as an antagonist of FliATd. TDE0201 is structurally similar to FlgM proteins, although its sequence is not conserved. Heterologous expression of TDE0201 in Escherichia coli inhibits its flagellin gene expression and motility. Biochemical and mutational analyses demonstrate that TDE0201 binds to FliATd and prevents it from binding to the σ28-dependent promoter. Deletions of flgM genes typically enhance bacterial class 3 flagellar gene expression; however, deletion of TDE0201 has an opposite effect (e.g., the mutant has a reduced level of flagellins). Follow-up studies revealed that deletion of TDE0201 leads to FliATd turnover, which in turn impairs the expression of flagellin genes. Swimming plate, cell tracking, and cryo-electron tomography analyses further disclosed that deletion of TDE0201 impairs spirochete motility and alters flagellar number and polarity: i.e., instead of having bipolar flagella, the mutant has flagella only at one end of cells. Collectively, these results indicate that TDE0201 is a FlgM homolog but acts differently from its counterparts in other bacteria. IMPORTANCE Spirochetes are a group of bacteria that cause several human diseases. A unique aspect of spirochetes is that they have bipolar periplasmic flagella (PFs), which bestow on the spirochetes a unique spiral shape and distinct swimming behaviors. While the structure and function of PFs have been extensively studied in spirochetes, the molecular mechanism that regulates the PFs' morphogenesis and assembly is poorly understood. In this report, FlgM, an anti-σ28 factor, is identified and functionally characterized in the oral spirochete Treponema denticola. Our results show that FlgM regulates the number and polarity of PFs via a unique mechanism. Identification of FliA and FlgM in T. denticola sets a benchmark to investigate their roles in other spirochetes.

Identification and Characterization of the Alternative σ28 Factor in Treponema denticola.

FliA (also known as σ28), a member of the bacterial σ70 family of transcription factors, directs RNA polymerase to flagellar late (class 3) promoters and initiates transcription. FliA has been studied in several bacteria, yet its role in spirochetes has not been established. In this report, we identify and functionally characterize a FliA homolog (TDE2683) in the oral spirochete Treponema denticola. Computational, genetic, and biochemical analyses demonstrated that TDE2683 has a structure similar to that of the σ28 of Escherichia coli, binds to σ28-dependent promoters, and can functionally replace the σ28 of E. coli. However, unlike its counterparts from other bacteria, TDE2683 cannot be deleted, suggesting its essential role in the survival of T. denticola. In vitro site-directed mutagenesis revealed that E221 and V231, two conserved residues in the σ4 region of σ28, are indispensable for the binding activity of TDE2683 to the σ28-dependent promoter. We then mutated these two residues in T. denticola and found that the mutations impair the expression of flagellin and chemotaxis genes and bacterial motility as well. Cryo-electron tomography analysis further revealed that the mutations disrupt the flagellar symmetry (i.e., number and placement) of T. denticola. Collectively, these results indicate that TDE2683 is a σ28 transcription factor that regulates the class 3 gene expression and controls the flagellar symmetry of T. denticola. To the best of our knowledge, this is the first report establishing the functionality of FliA in spirochetes. IMPORTANCE Spirochetes are a group of medically important but understudied bacteria. One of the unique aspects of spirochetes is that they have periplasmic flagella (PF, also known as endoflagella) which give rise to their unique spiral shape and distinct swimming behaviors and play a critical role in the pathophysiology of spirochetes. PF are structurally similar to external flagella, but the underpinning mechanism that regulates PF biosynthesis and assembly remains largely unknown. By using the oral spirochete Treponema denticola as a model, this report provides several lines of evidence that FliA, a σ28 transcriptional factor, regulates the late flagellin gene (class 3) expression, PF assembly, and flagellar symmetry as well, which provides insights into flagellar regulation and opens an avenue to investigate the role of σ28 in spirochetes.

Transcriptional and functional characterizations of multiple flagellin genes in spirochetes.

The flagellar filament is a helical propeller for bacterial locomotion. In external flagellates, the filaments are mostly homopolymers of a single flagellin protein. By contrast, the flagellar filaments of spirochetes are mostly heteropolymers of multiple flagellin proteins. This report seeks to investigate the role of multiple flagellin proteins using the oral spirochete Treponema denticola as a model. First, biochemical and genetic studies uncover that the flagellar filaments of T. denticola mainly comprise four proteins, FlaA, FlaB1, FlaB2, and FlaB3, in a defined stoichiometry. Second, transcriptional analyses reveal that the genes encoding these four proteins are regulated by two different transcriptional factors, sigma28 and sigma70 . Third, loss-of-function studies demonstrate that each individual flagellin protein contributes to spirochete motility, but none of them is absolutely required. Last, we provide genetic and structural evidence that FlaA forms a "seam"-like structure around the core and that deletion of individual flagellin protein alters the flagellar homeostasis. Collectively, these results demonstrate that T. denticola has evolved a unique mechanism to finely regulate its flagellar filament gene expression and assembly which renders the organelle with the right number, shape, strength, and structure for its distinct motility.

Genetic Manipulations of Oral Spirochete Treponema denticola.

There have been more than 60 different oral Treponema species identified in the oral cavity; however, only few species can be cultivated in vitro reliably. Among those cultivable species, due to its medical importance and genetic tractability, Treponema denticola, one of the keystone pathogens associated with human periodontitis, has emerged as a paradigm model organism to understanding the genetics, etiology, and pathophysiology of oral Treponema species. During the last two decades, several genetic tools have been developed, which have played an instrumental role in the study of T. denticola. This chapter describes the experimental design and procedure of genetic manipulations of T. denticola.

Atypical chemoreceptor arrays accommodate high membrane curvature.

The prokaryotic chemotaxis system is arguably the best-understood signaling pathway in biology. In all previously described species, chemoreceptors organize into a hexagonal (P6 symmetry) extended array. Here, we report an alternative symmetry (P2) of the chemotaxis apparatus that emerges from a strict linear organization of the histidine kinase CheA in Treponema denticola cells, which possesses arrays with the highest native curvature investigated thus far. Using cryo-ET, we reveal that Td chemoreceptor arrays assume an unusual arrangement of the supra-molecular protein assembly that has likely evolved to accommodate the high membrane curvature. The arrays have several atypical features, such as an extended dimerization domain of CheA and a variant CheW-CheR-like fusion protein that is critical for maintaining an ordered chemosensory apparatus. Furthermore, the previously characterized Td oxygen sensor ODP influences CheA ordering. These results suggest a greater diversity of the chemotaxis signaling system than previously thought.

Prevalence of phase variable epigenetic invertons among host-associated bacteria.

Type I restriction-modification (R-M) systems consist of a DNA endonuclease (HsdR, HsdM and HsdS subunits) and methyltransferase (HsdM and HsdS subunits). The hsdS sequences flanked by inverted repeats (referred to as epigenetic invertons) in certain Type I R-M systems undergo invertase-catalyzed inversions. Previous studies in Streptococcus pneumoniae have shown that hsdS inversions within clonal populations produce subpopulations with profound differences in the methylome, cellular physiology and virulence. In this study, we bioinformatically identified six major clades of the tyrosine and serine family invertases homologs from 16 bacterial phyla, which potentially catalyze hsdS inversions in the epigenetic invertons. In particular, the epigenetic invertons are highly enriched in host-associated bacteria. We further verified hsdS inversions in the Type I R-M systems of four representative host-associated bacteria and found that each of the resultant hsdS allelic variants specifies methylation of a unique DNA sequence. In addition, transcriptome analysis revealed that hsdS allelic variations in Enterococcus faecalis exert significant impact on gene expression. These findings indicate that epigenetic switches driven by invertases in the epigenetic invertons broadly operate in the host-associated bacteria, which may broadly contribute to bacterial host adaptation and virulence beyond the role of the Type I R-M systems against phage infection.

A di-iron protein recruited as an Fe[II] and oxygen sensor for bacterial chemotaxis functions by stabilizing an iron-peroxy species.

Many bacteria contain cytoplasmic chemoreceptors that lack sensor domains. Here, we demonstrate that such cytoplasmic receptors found in 8 different bacterial and archaeal phyla genetically couple to metalloproteins related to ß-lactamases and nitric oxide reductases. We show that this oxygen-binding di-iron protein (ODP) acts as a sensor for chemotactic responses to both iron and oxygen in the human pathogen Treponema denticola (Td). The ODP di-iron site binds oxygen at high affinity to reversibly form an unusually stable µ-peroxo adduct. Crystal structures of ODP from Td and the thermophile Thermotoga maritima (Tm) in the Fe[III]2-O22-, Zn[II], and apo states display differences in subunit association, conformation, and metal coordination that indicate potential mechanisms for sensing. In reconstituted systems, iron-peroxo ODP destabilizes the phosphorylated form of the receptor-coupled histidine kinase CheA, thereby providing a biochemical link between oxygen sensing and chemotaxis in diverse prokaryotes, including anaerobes of ancient origin.

A pleiotropic role of FlaG in regulating the cell morphogenesis and flagellar homeostasis at the cell poles of Treponema denticola.

FlaG homologue has been found in several bacteria including spirochetes; however, its function is poorly characterised. In this report, we investigated the role of TDE1473, a putative FlaG, in the spirochete Treponema denticola, a keystone pathogen of periodontitis. TDE1473 resides in a large gene operon that is controlled by a σ70 -like promoter and encodes a putative FlaG protein of 123 amino acids. TDE1473 can be detected in the periplasmic flagella (PFs) of T. denticola, suggesting that it is a flagella-associated protein. Consistently, in vitro studies demonstrate that the recombinant TDE1473 interacts with the PFs in a dose-dependent manner and that such an interaction requires FlaA, a flagellar filament sheath protein. Deletion of TDE1473 leads to long and less motile mutant cells. Cryo-electron tomography analysis reveal that the wild-type cells have 2-3 PFs with nearly homogenous lengths (ranging from 3 to 6 µm), whereas the mutant cells have less intact PFs with disparate lengths (ranging from 0.1 to 9 µm). The phenotype of T. denticola TDE1473 mutant reported here is different from its counterparts in other bacteria, which provides insight into further understanding the role of FlaG in the regulation of bacterial cell morphogenesis and flagellation.

A novel glycan modifies the flagellar filament proteins of the oral bacterium Treponema denticola.

While protein glycosylation has been reported in several spirochetes including the syphilis bacterium Treponema pallidum and Lyme disease pathogen Borrelia burgdorferi, the pertinent glycan structures and their roles remain uncharacterized. Herein, a novel glycan with an unusual chemical composition and structure in the oral spirochete Treponema denticola, a keystone pathogen of periodontitis was reported. The identified glycan of mass 450.2 Da is composed of a monoacetylated nonulosonic acid (Non) with a novel extended N7 acyl modification, a 2-methoxy-4,5,6-trihydroxy-hexanoyl residue in which the Non has a pseudaminic acid configuration (L-glycero-L-manno) and is ß-linked to serine or threonine residues. This novel glycan modifies the flagellin proteins (FlaBs) of T. denticola by O-linkage at multiple sites near the D1 domain, a highly conserved region of bacterial flagellins that interact with Toll-like receptor 5. Furthermore, mutagenesis studies demonstrate that the glycosylation plays an essential role in the flagellar assembly and motility of T. denticola. To our knowledge, this novel glycan and its unique modification sites have not been reported previously in any bacteria.

The Treponema denticola FhbB Protein Is a Dominant Early Antigen That Elicits FhbB Variant-Specific Antibodies That Block Factor H Binding and Cleavage by Dentilisin.

The Treponema denticola FhbB protein contributes to immune evasion by binding factor H (FH). Cleavage of FH by the T. denticola protease, dentilisin, may contribute to the local immune dysregulation that is characteristic of periodontal disease (PD). Although three FhbB phyletic types have been defined (FhbB1, FhbB2, and FhbB3), the in vivo expression patterns and antigenic heterogeneity of FhbB have not been assessed. Here, we demonstrate that FhbB is a dominant early antigen that elicits FhbB type-specific antibody (Ab) responses. Using the murine skin abscess model, we demonstrate that the presence or absence of FhbB or dentilisin significantly influences Ab responses to infection and skin abscess formation. Competitive binding analyses revealed that α-FhbB Ab can compete with FH for binding to T. denticola and block dentilisin-mediated FH cleavage. Lastly, we demonstrate that dentilisin cleavage sites reside within critical functional domains of FH, including the complement regulatory domain formed by CCPs 1 to 4. Analysis of the FH cleavage products revealed that they lack cofactor activity. The data presented here provide insight into the in vivo significance of dentilisin, FhbB and its antigenic diversity, and the potential impact of FH cleavage on the regulation of complement activation.

pyrF as a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola.

The pathophysiology of Treponema denticola, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate that pyrF, a gene encoding orotidine-5'-monophosphate decarboxylase, can be used as a counterselectable marker in T. denticola to construct marker-free mutants. T. denticola is susceptible to 5-fluoroorotic acid (5-FOA). To establish a pyrF-based counterselectable knockout system in T. denticola, the pyrF gene was deleted. The deletion conferred resistance to 5-FOA in T. denticola. Next, a single-crossover mutant was constructed by reintroducing pyrF along with a gentamicin resistance gene (aacC1) back into the chromosome of the pyrF mutant at the locus of choice. In this study, we chose flgE, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. The obtained single-crossover mutant (named FlgE(in)) regained the susceptibility to 5-FOA. Finally, FlgE(in) was plated on solid agar containing 5-FOA. Numerous colonies of the 5-FOA-resistant mutant (named FlgE(out)) were obtained and characterized by PCR and Southern blotting analyses. The results showed that the flgE gene was deleted and FlgE(out) was free of selection markers (i.e., pyrF and aacC1). Compared to previously constructed flgE mutants that contain an antibiotic selection marker, the deletion of flgE in FlgE(out) has no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity of T. denticola.

A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochaete Treponema denticola.

Neuraminidases (sialidases) catalyse the removal of terminal sialic acid from glycoconjugates. Bacterial pathogens often utilize neuraminidases to scavenge host sialic acid, which can be utilized either as a nutrient or as a decorating molecule to disguise themselves from host immune attacks. Herein, a putative neuraminidase (TDE0471) was identified in Treponema denticola, an oral spirochaete associated with human periodontitis. TDE0471 is a cell surface-exposed exo-neuraminidase that removes sialic acid from human serum proteins; it is required for T.denticola to grow in a medium that mimics gingival crevice fluid, suggesting that the spirochaete may use sialic acid as a nutrient in vivo. TDE0471 protects T.denticola from serum killing by preventing the deposition of membrane attack complexes on the bacterial cell surface. Animal studies revealed that a TDE0471-deficient mutant is less virulent than its parental wild-type strain in BALB/C mice. However, it causes a level of tissue damage similar to the wild type in complement-deficient B6.129S4-C3(tm1) (Crr) /J mice albeit the damage caused by both bacterial strains is more severe in these transgenic mice. Based on these results, we propose that T.denticola has evolved a strategy to scavenge host sialic acid using its neuraminidase, which allows the spirochaete to acquire nutrients and evade complement killing.