Effect of WQ-3334 on Campylobacter jejuni carrying a DNA gyrase with dominant amino acid substitutions conferring quinolone resistance.

INTRODUCTION: Campylobacteriosis stands as one of the most frequent bacterial gastroenteritis worldwide necessitating antibiotic treatment in severe cases and the rise of quinolones-resistant Campylobacter jejuni poses a significant challenge. The predominant mechanism of quinolones-resistance in this bacterium involves point mutations in the gyrA, resulting in amino acid substitution from threonine to isoleucine at 86th position, representing more than 90% of mutant DNA gyrase, and aspartic acid to asparagine at 90th position. WQ-3334, a novel quinolone, has demonstrated strong inhibitory activity against various bacteria. This study aims to investigate the effectiveness of WQ-3334, and its analogues, WQ-4064 and WQ-4065, with a unique modification in R1 against quinolones-resistant C. jejuni. METHODS: The structure-activity relationship of the examined drugs was investigated by measuring IC50 and their antimicrobial activities were accessed by MIC against C. jejuni strains. Additionally, in silico docking simulations were carried out using the crystal structure of the Escherichia coli DNA gyrase. RESULT: WQ-3334 exhibited the lowest IC50 against WT (0.188 ± 0.039 mg/L), T86I (11.0 ± 0.7 mg/L) and D90 N (1.60 ± 0.28 mg/L). Notably, DNA gyrases with T86I substitutions displayed the highest IC50 values among the examined WQ compounds. Moreover, WQ-3334 demonstrated the lowest MICs against wild-type and mutant strains. The docking simulations further confirmed the interactions between WQ-3334 and DNA gyrases. CONCLUSION: WQ-3334 with 6-amino-3,5-difluoropyridine-2-yl at R1 severed as a remarkable candidate for the treatment of foodborne diseases caused by quinolones-resistant C. jejuni as shown by the high inhibitory activity against both wild-type and the predominant quinolones-resistant strains.

Characterization of HcaA, a novel autotransporter protein in Helicobacter cinaedi, and its role in host cell adhesion.

IMPORTANCE: Helicobacter species are classified as gastric or enterohepatic according to their habitat. Among enterohepatic Helicobacter species, which inhabit the intestine, colon, and liver, Helicobacter cinaedi has been most frequently isolated from humans. H. cinaedi often causes bacteremia and cellulitis in immunocompromised hosts. Here, we focused on the H. cinaedi autotransporter protein A (HcaA), a novel virulence factor in H. cinaedi. We discovered that HcaA contributes to cell adhesion via its Arg-Gly-Asp motif. Furthermore, in animal experiments, bacterial colonization was reduced in mice infected with HcaA-knockout strains, supporting the hypothesis that HcaA contributes to H. cinaedi adhesion to host cells. Our study provides a novel mechanism for the establishment of H. cinaedi infections and provides new insights into the role of autotransporter proteins in the establishment of Helicobacter infection.

Complete genome sequences of nine Burkholderia pseudomallei strains.

We report the complete genome sequences of nine Burkholderia pseudomallei strains preserved in research facilities in Japan; GTC3P0019, GTC3P0050, GTC3P0054, GTC3P0254T (type strain), Kanagawa, Tokushima, KM376, KM390, and KM391. The genomic information of these strains may provide references for comparative studies of B. pseudomallei pathogenicity.

Molecular Characterization of Mycobacterium ulcerans DNA Gyrase and Identification of Mutations Reducing Susceptibility to Quinolones In Vitro.

Buruli ulcer disease is a neglected necrotizing and disabling cutaneous tropical illness caused by Mycobacterium ulcerans. Fluoroquinolone (FQ), used in the treatment of this disease, has been known to act by inhibiting the enzymatic activities of DNA gyrase. However, the detailed molecular basis of these characteristics and the FQ resistance mechanisms in M. ulcerans remains unknown. This study investigated the detailed molecular mechanism of M. ulcerans DNA gyrase and the contribution of FQ resistance in vitro using recombinant proteins from the M. ulcerans subsp. shinshuense and Agy99 strains with reduced sensitivity to FQs. The IC50 of FQs against Ala91Val and Asp95Gly mutants of M. ulcerans shinshuense and Agy99 GyrA subunits were 3.7- to 42.0-fold higher than those against wild-type (WT) enzyme. Similarly, the quinolone concentrations required to induce 25% of the maximum DNA cleavage (CC25) was 10- to 210-fold higher than those for the WT enzyme. Furthermore, the interaction between the amino acid residues of the WT/mutant M. ulcerans DNA gyrase and FQ side chains were assessed by molecular docking studies. This was the first elaborative study demonstrating the contribution of mutations in M. ulcerans DNA GyrA subunit to FQ resistance in vitro.

Interaction of Quinolones Carrying New R1 Group with Mycobacterium leprae DNA Gyrase.

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and the treatment of choice is ofloxacin (OFX). Specific amino acid substitutions in DNA gyrase of M. leprae have been reported leading to resistance against the drug. In our previous study, WQ-3810, a fluoroquinolone with a new R1 group (6-amino-3,5-difluoropyridin-2-yl) was shown to have a strong inhibitory activity on OFX-resistant DNA gyrases of M. leprae, and the structural characteristics of its R1 group was predicted to enhance the inhibitory activity. Methodology/Principal Finding: To further understand the contribution of the R1 group, WQ-3334 with the same R1 group as WQ-3810, WQ-4064, and WQ-4065, but with slightly modified R1 group, were assessed on their activities against recombinant DNA gyrase of M. leprae. An in silico study was conducted to understand the molecular interactions between DNA gyrase and WQ compounds. WQ-3334 and WQ-3810 were shown to have greater inhibitory activity against M. leprae DNA gyrase than others. Furthermore, analysis using quinolone-resistant M. leprae DNA gyrases showed that WQ-3334 had greater inhibitory activity than WQ-3810. The R8 group was shown to be a factor for the linkage of the R1 groups with GyrB by an in silico study. Conclusions/Significance: The inhibitory effect of WQ compounds that have a new R1 group against M. leprae DNA gyrase can be enhanced by improving the binding affinity with different R8 group molecules. The information obtained by this work could be applied to design new fluoroquinolones effective for quinolone-resistant M. leprae and other bacterial pathogens.

Immunodominant proteins P1 and P40/P90 from human pathogen Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections.

Truncated Pneumolysin from Streptococcus pneumoniae as a TLR4-Antagonizing New Drug for Chronic Inflammatory Conditions.

Microbial proteins have recently been found to have more benefits in clinical disease treatment because of their better-developed strategy and properties than traditional medicine. In this study, we investigated the effectiveness of a truncated peptide synthesized from the C-terminal sequence of pneumolysin, i.e., C70PLY4, in Streptococcus pneumoniae, in treating chronic inflammatory conditions. It has been shown that C70PLY4 significantly blocks the transendothelial migration of neutrophils and attenuates the formation of atherosclerotic plaque and the secretion of soluble forms of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in high-fat-diet/streptozotocin-induced inflammatory rats. The mechanism and the docking simulation analysis further indicated that C70PLY4 might serve as a Toll-like receptor 4 (TLR4) antagonist by competing for the binding site of MD2, an indispensable protein for lipopolysaccharide (LPS)-TLR4 interaction signaling, on the TLR4 structure. Moreover, compared to the full-length PLY, C70PLY4 seems to have no cytotoxicity in human vascular endothelial cells. Our study elucidated a possible therapeutic efficacy of C70PLY4 in reducing chronic inflammatory conditions and clarified the underlying mechanism. Thus, our findings identify a new drug candidate that, by blocking TLR4 activity, could be an effective treatment for patients with chronic inflammatory diseases.

Cloning and Protein Expression of eccB5 Gene in ESX-5 System from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis in human. One of the major M. tuberculosis virulence factors is early secretory antigenic target of 6-kDa (ESAT-6), and EccB5 protein encoded by eccB5 is one of its components. EccB5 protein is a transmembrane protein in ESX-5 system. The aim of this study is to explore the characteristics of wild-type EccB5 and its mutant form N426I. We expressed the EccB5 protein by cloning the mutant and wild-type eccB5 gene in Escherichia coli (E. coli). We compared the protein structure of wild type and mutant form of EccB5 and found changes in structure around Asn426 (loop structure) in wild type and around Ile426 (ß-strand) in the mutant. The truncated recombinant protein of EccB5 was successfully cloned and expressed using plasmid pCold I in E. coli DH5α and E. coli strain Rosetta-gami B (DE3) and purified as a 38.6 kDa protein by using the affinity column. There was no detectable adenosine triphosphatase activity in truncated forms of EccB5 and its mutant. In conclusion, our study reveals successful cloning and protein expression of truncated form of eccB5 gene of M. tuberculosis. EccB5 protein in ESX-5 system may be an important membrane component involved in the transport machinery of type VII secretion system, which is essential for growth and virulence.

Complete Genome Sequence of Helicobacter suis Strain SNTW101c, Originally Isolated from a Patient with Nodular Gastritis.

Helicobacter suis strain SNTW101c, which was originally obtained from a patient with nodular gastritis, has been maintained in mouse stomach because of difficulty culturing it in vitro Recently, we succeeded in culturing this strain in vitro Here, we report the complete genome sequence of H. suis strain SNTW101c.

WQ-3810 inhibits DNA gyrase activity in ofloxacin-resistant Mycobacterium leprae.

BACKGROUND: Mycobacterium leprae causes leprosy and ofloxacin is used to control this bacterium. However, specific amino acid substitutions in DNA gyrases of M. leprae interferes with the effect of ofloxacin. METHODOLOGY/PRINCIPAL FINDINGS: Here we tested the inhibitory effect of WQ-3810 on DNA gyrases in M. leprae, using recombinant gyrases. We theorized that WQ-3810 and DNA gyrases interacted, which was tested in silico. Compared with control drugs like ofloxacin, WQ-3810 showed a better inhibitory effect on ofloxacin-resistant DNA gyrases. The in-silico study showed that, unlike control drugs, a specific linkage between a R1 group in WQ-3810 and aspartic acid at position 464 in the subunit B of DNA gyrases existed, which would enhance the inhibitory effect of WQ-3810. This linkage was confirmed in a further experiment, using recombinant DNA gyrases with amino acid substitutions in subunits B instead. CONCLUSIONS/SIGNIFICANCE: The inhibitory effect of WQ-3810 was likely enhanced by the specific linkage between a R1 group residue in its structure and DNA gyrases. Using interactions like the one found in the present work may help design new fluoroquinolones that contribute to halt the emergence of antibiotic-resistant pathogens.

DNA gyrase could be a crucial regulatory factor for growth and survival of Mycobacterium leprae.

Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in vitro, except in mouse footpads in vivo. The molecular basis of these characteristics and the mechanisms remain unknown. Consequently, analysis of Mle growth and survival is urgently needed to develop novel therapies against leprosy, including rapid, simple, and specific methods to detect infection. Here, we demonstrated the functional role and contribution of Mle-DNA gyrase, which regulates DNA topology, DNA replication, and chromosome segregation to promote bacterial growth and survival, in Mle growth and survival in vitro and in vivo. The optimum temperature for Mle-DNA gyrase activity was 30 °C. When the DNA gyrB-gyrA genes in Mycobacterium smegmatis were replaced with the Mle gyrase genes by allelic exchange, the recombinants could not grow at 37 °C. Moreover, using radiorespirometry analysis for viability of Mle bacilli, we found that Mle growth was more vigorous at 25-30 °C than at 37 °C, but was inhibited above 40 °C. These results propose that DNA gyrase is a crucial factor for Mle growth and survival and its sensitivity to temperature may be exploited in heat-based treatment of leprosy.

Production and characterization of recombinant P1 adhesin essential for adhesion, gliding, and antigenic variation in the human pathogenic bacterium, Mycoplasma pneumoniae.

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6 × His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.

Mutations in Genes Encoding Penicillin-Binding Proteins and Efflux Pumps Play a Role in ß-Lactam Resistance in Helicobacter cinaedi.

ß-Lactams are often used to treat Helicobacter cinaedi infections; however, the mechanism underlying ß-lactam resistance is unknown. In this study, we investigated ß-lactam resistance in an H. cinaedi strain, MRY12-0051 (MICs of amoxicillin [AMX] and ceftriaxone [CRO], 32 and 128 µg/ml; obtained from human feces). Based on a comparative whole-genome analysis of MRY12-0051 and the CRO-susceptible H. cinaedi strain MRY08-1234 (MICs of AMX and CRO, 1 and 4 µg/ml; obtained from human blood), we identified five mutations in genes encoding penicillin-binding proteins (PBPs), including two in pbpA, one in pbp2, and two in ftsI Transformation and penicillin binding assays indicated that CRO resistance was mainly associated with mutations in pbpA; mutations in ftsI also led to increased resistance to AMX. Knocking out cmeB and cmeD, which encode resistance-nodulation-division-type efflux pump components, in H. cinaedi type strain CCUG18818 (AMX MIC, 4 to 8 µg/ml) resulted in 8- and 64-fold decreases, respectively, in the AMX MIC. Hence, MICs of AMX in H. cinaedi become similar to those of Helicobacter pylori isolates in the absence of cmeD In conclusion, the difference in susceptibility to ß-lactams between H. pylori and H. cinaedi is explained by differences in efflux pump components. Mutations in pbpA are the primary determinant of high resistance to ß-lactams in H. cinaedi.

Biochemical and structural investigations on phosphoribosylpyrophosphate synthetase from Mycobacterium smegmatis.

Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest.

Structural Insights into Recognition of Hydrolyzed Carbapenems and Inhibitors by Subclass B3 Metallo-ß-Lactamase SMB-1.

Metallo-ß-lactamases (MBLs) confer resistance to carbapenems, and their increasing global prevalence is a growing clinical concern. To elucidate the mechanisms by which these enzymes recognize and hydrolyze carbapenems, we solved 1.4 to 1.6 Å crystal structures of SMB-1 (Serratia metallo-ß-lactamase 1), a subclass B3 MBL, bound to hydrolyzed carbapenems (doripenem, meropenem, and imipenem). In these structures, SMB-1 interacts mainly with the carbapenem core structure via elements in the active site, including a zinc ion (Zn-2), Q157[113] (where the position in the SMB-1 sequence is in brackets after the BBL number), S221[175], and T223[177]. There is less contact with the carbapenem R2 side chains, strongly indicating that SMB-1 primarily recognizes the carbapenem core structure. This is the first report describing how a subclass B3 MBL recognizes carbapenems. We also solved the crystal structure of SMB-1 in complex with the approved drugs captopril, an inhibitor of the angiotensin-converting enzyme, and 2-mercaptoethanesulfonate, a chemoprotectant. These drugs are inhibitors of SMB-1 with Ki values of 8.9 and 184 µM, respectively. Like carbapenems, these inhibitors interact with Q157[113] and T223[177] and their thiol groups coordinate the zinc ions in the active site. Taken together, the data indicate that Q157[113], S221[175], T223[177], and the two zinc ions in the active site are key targets in the design of SMB-1 inhibitors with enhanced affinity. The structural data provide a solid foundation for the development of effective inhibitors that would overcome the carbapenem resistance of MBL-producing multidrug-resistant microbes.

Mycobacterium avium infection induces the resistance of the interferon-γ response in mouse spleen cells at late stages of infection.

BACKGROUND: Bacterial infections cause an increase in the population of hematopoietic stem cells (HSCs). To investigate the downstream factors associated with hematopoietic stem cells, mice are infected with Mycobacterium avium (M. avium). RESULTS: Mycobacterium avium (M. avium) infection induces the enlargement of the spleen and changes in histopathology, including changes to the lineage populations. A dramatic expansion of Lin-c-kit+Sca-1+ (KSL) cells in mouse bone marrow cells and spleen cells was detected 4 weeks after infection with M. avium; however, there was no difference in the engraft activity between infected and un-infected mouse bone marrow cells. We tested the cytokine and cytokine-related gene expression after M. avium infection and found that IFN-γ expression increased and peaked at 4 weeks in both bone marrow and spleen cells. The expression of Sca-1 gene peaked at 4 weeks in the bone marrow but peaked at 2 weeks in spleen cells, although the Sca-1 surface marker peaked at 4 weeks after infection in both bone marrow and spleen cells. Interferon regulatory factor-2 (IRF-2) expression did not change in the bone marrow cells, whereas it decreased in spleen cells at 4 weeks and IRF-1 expression was up-regulated in both bone marrow and spleen cells after infection. However, the up-regulation of IRF-1 was not correlated with IFN-γ expression in the M. avium-infected mouse spleen cells. CONCLUSIONS: This finding suggests that the IFN-γ production mediated by M. avium infection alters the population of KSL cells during host defense, and the down-regulation of the IFN-γ response in spleen cells occurs at the late stage after M. avium infection.

The Other Helicobacters.

In the past year, a substantial number of (putative) novel Helicobacter species have been described, including Helicobacter himalayensis colonizing the Himalayan marmot and Helicobacter apodemus, colonizing the Korean striped field mouse. In addition, a putative novel gastric Helicobacter species was identified in wild gorillas and chimpanzees, for which the name "Candidatus H. homininae" was proposed. A high incidence of gastric non-H. pylori Helicobacter infection was described in China and multiple case reports have described the involvement of enterohepatic Helicobacter species, especially Helicobacter cinaedi, in a wide range of diseases. Several studies in rodent models further elucidated the mechanisms underlying the development of gastric mucosa-associated lymphoid tissue lymphoma during infection with gastric non-H. pylori Helicobacters. The effects of infection with gastric Helicobacters on the development of neuroinflammation were investigated and several enterohepatic Helicobacter species were shown to affect the composition of the gut microbiota, to influence vaccine efficiency as well as the progression of cancer in distant sites of the body.

Purification and functional characterization of diadenosine 5',5‴-P(1),P(4)-tetraphosphate phosphorylases from Mycobacterium smegmatis and Mycobacterium avium.

We recently demonstrated that the Rv2613c protein from Mycobacterium tuberculosis H37Rv is a novel diadenosine 5',5‴-P(1),P(4)-tetraphosphate (Ap4A) phosphorylase (MtAPA) that forms a tetramer. Mycobacterium avium and Mycobacterium smegmatis express proteins named MAV_3489 and MSMEG_2932, respectively, that are homologous to MtAPA. Here we showed that the MAV_3489 and MSMEG_2932 proteins possess Ap4A phosphorylase activity and enzymatic properties similar to those of MtAPA. Furthermore, gel-filtration column chromatography revealed that MAV_3489 and MSMEG_2932 assembled into homotetramers in solution, indicating that they may also form unique Ap4A-binding sites composed of tetramers.

Roles of Ala-149 in the catalytic activity of diadenosine tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv.

Diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap4A) phosphorylase from Mycobacterium tuberculosis H37Rv (MtAPA) belongs to the histidine triad motif (HIT) superfamily, but is the only member with an alanine residue at position 149 (Ala-149). Enzymatic analysis revealed that the Ala-149 deletion mutant displayed substrate specificity for diadenosine 5',5'''-P(1),P(5)-pentaphosphate and was inactive on Ap4A and other substrates that are utilized by the wild-type enzyme.