VirB4- and VirD4-Like ATPases, Components of a Putative Type 4C Secretion System in Clostridioides difficile

Abstract

The type 4 secretion system (T4SS) represents a bacterial nanomachine capable of trans-cell wall transportation of proteins and DNA and has attracted intense interest due to its roles in the pathogenesis of infectious diseases. In the current investigation, we uncovered three distinct gene clusters in Clostridioides difficile strain 630 encoding proteins structurally related to components of the VirB4/D4 type 4C secretion system from Streptococcus suis strain 05ZYH33 and located within sequences of conjugative transposons (CTn). Phylogenic analysis revealed that VirB4- and VirD4-like proteins of the CTn4 locus, on the one hand, and those of the CTn2 and CTn5 loci, on the other hand, fit into separate clades, suggesting specific roles of identified secretion system variants in the physiology of C. difficile . Our further study on VirB4- and VirD4-like products encoded by CTn4 revealed that both proteins possess Mg 2+ -dependent ATPase activity, form oligomers (most likely hexamers) in aqueous solutions, and rely on potassium but not sodium ions for the highest catalytic rate. VirD4 binds nonspecifically to DNA and RNA. The DNA-binding activity of VirD4 strongly decreased with the W241A variant. Mutations in the nucleotide sequences encoding presumable Walker A and Walker B motifs decreased the stability of the oligomers and significantly but not completely attenuated the enzymatic activity of VirB4. In VirD4, substitutions of amino acid residues in the peptides reminiscent of Walker structural motifs neither attenuated the enzymatic activity of the protein nor influenced the oligomerization state of the ATPase. Importance C. difficile is a gram-positive, anaerobic, spore-forming bacterium that causes life-threatening colitis in humans. Major virulence factors of the microorganism include the toxins TcdA, TcdB and CDT. However, other bacterial products, including a type 4C secretion system, have been hypothesized to contribute to the pathogenesis of the infection and are considered possible virulence factors of C. difficile . In the current paper, we describe the structural organization of putative T4SS machinery in C. difficile and characterize its VirB4- and VirD4-like components. Our studies, in addition to its significance for basic science, can potentially aid the development of antivirulence drugs suitable for the treatment of C. difficile infection.

Genome sequence and characterization of a hypervirulent BI/NAP1/027Clostridioides difficile(CDC20121308)

“…According to the classification of Zhang and collaborators, this GI corresponds to a new conjugative transposon 5-like (CTn5-like) (Zhang et al, 2017). Moreover, we identified VirD4, VirB4, and VirB6-like coding sequences similar to what was previously reported for the 630 strain (Sorokina et al, 2021). BLASTp of CDC20121308 VirD, VirB4 and VirB6-like protein sequences against C. difficile from NCBI database showed 100% homology with many strains, suggesting a conserved core of the type IVC secretory system (data not shown).…”

Whole-Genome Analysis of Acinetobacter baumannii Strain AB43 Containing a Type I-Fb CRISPR-Cas System: Insights into the Relationship with Drug Resistance

“…Several studies also show that CRISPR-Cas systems have an important role in virulence regulation in multiple pathogenic bacteria [ 47 ]. VirB4, as a virulence protein of pathogenic bacteria, is a key component of VirB secretion apparatus and has high similarity with members of the type IV secretion system [ 48 ]. Two specific spacers matched to VirB4 of plasmids present in the AB43 CRISPR array may influence the virulence of this strain.…”

Type 4 secretion system in Clostridioides difficile: Structural features and its role as a pathogenicity factor

“…До сих пор не определён точный состав секреторного аппарата. Помимо ранее выделенных генов VirD4, VirB6 и VirB4 [24] с использованием биоинформационного анализа мы обнаружили ген, предположительно кодирующий гомолог VirB1 во всех трех конъюгативных транспозонах: CTn4, CTn2 и CTn5 (последние обозначены нами в данной статье CTn2/CTn5) [31]. В состав конъюгативной части оперонов CTn у C. difficile также входят гены метилтрансферазы, релаксазы, хеликазы и топоизомеразы.…”

“…[27]. В то же время связь СС4Т с патогенезом заболеваний, вызываемых грамположительными возбудителями, только начинает привлекать внимание микробиологов [28][29][30][31]. Уже сейчас становится ясно, что изучение структуры и функции секретор-ного аппарата этой группы бактерий представляется важным не только для расшифровки инфекционных процессов, но и для создания средств терапии и профилактики заболеваний [28,30].…”