Zhukhovitsky, V. G. | Search bioRxiv

bioRxiv2021

VirB4- and VirD4-like ATPases, components of a putative type 4C secretion system in Clostridioides difficile

The type 4 secretion system (T4SS) represents a bacterial nanomachine capable of trans-cell wall transportation of proteins and DNA and which has attracted intense interest due to its roles in the pathogenesis of infectious diseases. During the current investigation we uncovered three distinct gene clusters in Clostridioides difficile strain 630 coding for 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 shows that VirB4- and VirD4-like proteins of CTn4 locus, on one hand, and those of CTn2 and CTn5 loci, on the other hand, fit into separate clades, suggesting specific roles of identified secretion system variants in physiology of C. difficile. Our further study on VirB4- and VirD4-like products coded by CTn4 revealed that both proteins possess Mg2+-dependent ATPase activity, form oligomers (most probably, hexamers) in water solutions, and rely on potassium but not sodium ions for the highest catalytic rate. VirD4 binds nonspecifically to DNA and RNA. Its DNA binding activity strongly decreased with the W241A variant. Mutations in the nucleotide sequences coding for presumable Walker A and Walker B motifs decreased stability of the oligomers and significantly but not completely attenuated enzymatic activity of VirB4. In VirD4, substitutions of amino acid residues in the peptides reminiscent of Walker structural motifs resulted neither in attenuation of enzymatic activity of the protein nor influenced the oligomerization state of the ATPase. ImportanceC. difficile is a Gram-positive, anaerobic, spore-forming bacterium that causes life-threatening colitis in humans. Major virulence factors of the microorganism include 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 as possible virulence factors of C. difficile. In the current paper we describe structural organization of putative T4SS machinery in C. difficile and characterize its VirB4- and VirD4-like components. Our studies, in addition to significance for basic science, can potentially aid development of anti-virulence drugs suitable for treatment of C. difficile infection.

microbiology↗

bioRxiv2019

Microcolonies: a novel morphological form of pathogenic Mycoplasma spp

The work describes a novel morphological form found in 5 species of Mollicutes: Mycoplasma hominis, M. fermentans, M. gallisepticum, M. pneumoniae, Acholeplasma laidlawii. The form, which is referred to as microcolonies (MCs) in contrast to typical colonies (TCs), was characterized by tiny propeller-shaped colonies formed by rod-like cells tightly packed in parallel rows. MCs were observed within routinely cultivated type strain cultures of the listed species, and rod-like cells could be seen ewith SEM within TCs of the "fried-egg" type. Pure MC cultures were obtained by treatment of Mycoplasma cultures with hyperimmune serum, antibiotics or argon non-thermal plasma. Appearance of MCs was observed 7-12 days post plating while TCs appeared 24-48 h post plating. MCs derived from the M. hominis type strain H-34 were characterized in details. MCs did not differ from the parental culture in the MLST, direct fluorescent and epifluorescent tests and Western-blotting with a monospecific camel-derived nanoantibody aMh-FcG2a. Meanwhile, MCs derived from this strain and other listed species were resistant to at least 9 antibiotics and did not hydrolyze arginine and/or glucose in standard bacteriological tests. MC cultures that belonged to M. hominis (n=70), M. pneumoniae (n=2), M. fermentans (n=2), Mycoplasma spp (n=5) were isolated from clinical samples of serum, synovial liquid and urina of patients with inflammatory urogenital tract diseases, asthma, arthritis. The reported MCs might be similar to "small colony variants" (SCVs) described in other bacterial species. However, in contrast to SCVs, MCs have never reverted to TCs. Multiple consecutive re-plating steps (for up to 3 years) were not sufficient to provide appearance of TCs within a pure MC culture. An unknown role of MCs in infection pathology along with their prominent antibiotic resistance makes them a challenge for the future studies of Mollicutes. Author summaryHere we demonstrated that Mycoplasma species form small size colonies (referred to as minicolonies, MCs). MC size is ten times less than the size of typical Mycoplasma colonies (TCs). MCs are very slow growing: it was required 9-10 days for MCs to form in contrast to 24-72 h required for TCs to form. The presents a system of evidences that MCs are formed by the same species as TCs, which they have been obtained from. Pure culture of MCs might be obtained from TC cultures by treatment with the hyperimmune serum, antibiotic and non-thermal gas plasma. MCs of all species were resistant to antibiotics effective against TCs. MCs did not hydrolyze arginine and glucose in standard bacteriological tests. MCs of different Mycoplasma species were isolated from clinical samples of sera, urea and synovial fluids from patients with urolithiasis, rheumatoid arthritis and asthma. MCs never have reverted to TCs even after three years passing. A role of MCs in infectious pathology has not been established yet. Nevertheless, ability to persist in the human body and extreme antibiotic resistance make MCs to be a challenge for the future research.

microbiology↗