Development of novel isatin-nicotinohydrazide hybrids with potent activity against susceptible/resistant Mycobacterium tuberculosis and bronchitis causing-bacteria.

Joining the global fight against Tuberculosis, the world's most deadly infectious disease, herein we present the design and synthesis of novel isatin-nicotinohydrazide hybrids (5a-m and 9a-c) as promising anti-tubercular and antibacterial agents. The anti-tubercular activity of the target hybrids was evaluated against drug-susceptible M. tuberculosis strain (ATCC 27294) where hybrids 5d, 5g and 5h were found to be as potent as INH with MIC = 0.24 µg/mL, also the activity was evaluated against Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823) where compounds 5g and 5h showed excellent activity (MIC = 3.9 µg/mL). Moreover, the target hybrids were examined against six bronchitis causing-bacteria. Most derivatives exhibited excellent antibacterial activity. K. pneumonia emerged as the most sensitive strain with MIC range: 0.49-7.81 µg/mL. Furthermore, a molecular docking study has proposed DprE1 as a probable enzymatic target for herein reported isatin-nicotinohydrazide hybrids, and explored the binding interactions within the vicinity of DprE1 active site.

Biophysical and In-Silico Studies of Phytochemicals Targeting Chorismate Synthase from Drug-Resistant Moraxella Catarrhalis.

Chorismate serves as a crucial precursor for the synthesis of many aromatic compounds essential for the survival and virulence in various bacteria and protozoans. Chorismate synthase, a vital enzyme in the shikimate pathway, is responsible for the formation of chorismate from enolpyruvylshikimate-3-phosphate (EPSP). Moraxella catarrhalis is reported to be resistant to many beta-lactam antibiotics and causes chronic ailments such as otitis media, sinusitis, laryngitis, and bronchopulmonary infections. Here, we have cloned the aroC gene from Moraxella catarrhalis in pET28c and heterologously produced the chorismate synthase (~ 43 kDa) in Escherichia coli BL21(DE3) cells. We have predicted the three-dimensional structure of this enzyme and used the refined model for ligand-based virtual screening against Supernatural Database using PyRx tool that led to the identification of the top three molecules (caffeic acid, gallic acid, and o-coumaric acid). The resultant protein-ligand complex structures were subjected to 50 ns molecular dynamics (MD) simulation using GROMACS. Further, the binding energy was calculated by MM/PBSA approach using the trajectory obtained from MD simulation. The binding affinities of these compounds were validated with ITC experiments, which suggest that gallic acid has the highest binding affinity amongst these three phytochemicals. Together, these results pave the way for the use of these phytochemicals as potential anti-bacterial compounds.

The Moraxella catarrhalis phase-variable DNA methyltransferase ModM3 is an epigenetic regulator that affects bacterial survival in an in vivo model of otitis media.

BACKGROUND: Moraxella catarrhalis is a leading cause of otitis media (OM) and chronic obstructive pulmonary disease (COPD). M. catarrhalis contains a Type III DNA adenine methyltransferase (ModM) that is phase-variably expressed (i.e., its expression is subject to random, reversible ON/OFF switching). ModM has six target recognition domain alleles (modM1-6), and we have previously shown that modM2 is the predominant allele, while modM3 is associated with OM. Phase-variable DNA methyltransferases mediate epigenetic regulation and modulate pathogenesis in several bacteria. ModM2 of M. catarrhalis regulates the expression of a phasevarion containing genes important for colonization and infection. Here we describe the phase-variable expression of modM3, the ModM3 methylation site and the suite of genes regulated within the ModM3 phasevarion. RESULTS: Phase-variable expression of modM3, mediated by variation in length of a 5'-(CAAC)n-3' tetranucleotide repeat tract in the open reading frame was demonstrated in M. catarrhalis strain CCRI-195ME with GeneScan fragment length analysis and western immunoblot. We determined that ModM3 is an active N6-adenine methyltransferase that methylates the sequence 5'-ACm6ATC-3'. Methylation was detected at all 4446 5'-ACATC-3' sites in the genome when ModM3 is expressed. RNASeq analysis identified 31 genes that are differentially expressed between modM3 ON and OFF variants, including five genes that are involved in the response to oxidative and nitrosative stress, with potential roles in biofilm formation and survival in anaerobic environments. An in vivo chinchilla (Chinchilla lanigera) model of otitis media demonstrated that transbullar challenge with the modM3 OFF variant resulted in an increased middle ear bacterial load compared to a modM3 ON variant. In addition, co-infection experiments with NTHi and M. catarrhalis modM3 ON or modM3 OFF variants revealed that phase variation of modM3 altered survival of NTHi in the middle ear during early and late stage infection. CONCLUSIONS: Phase variation of ModM3 epigenetically regulates the expression of a phasevarion containing multiple genes that are potentially important in the progression of otitis media.

Biochemical and biophysical characterization of 1,4-naphthoquinone as a dual inhibitor of two key enzymes of type II fatty acid biosynthesis from Moraxella catarrhalis.

The fatty acid biosynthesis (FAS II) is a vital process in bacteria and regarded as an attractive pathway for the development of potential antimicrobial agents. In this study, we report 1,4-naphthoquinone (NPQ) as a dual inhibitor of two key enzymes of FAS II pathway, namely FabD (Malonyl-CoA:ACP transacylase) and FabZ (ß-hydroxyacyl-ACP dehydratase). Mode of inhibition of NPQ was found to be non-competitive for both enzymes with IC50 of 26.67 µΜ and 23.18 µΜ against McFabZ and McFabD respectively. Conformational changes in secondary and tertiary structures marked by the loss of helical contents were observed in both enzymes upon NPQ binding. The fluorescence quenching was found to be static with a stable ground state complex formation. ITC based studies have shown that NPQ is binding to McFabZ with a stronger affinity (~1.5×) as compared to McFabD. Molecular docking studies have found that NPQ interacts with key residues of both McFabD (Ser209, Arg126, and Leu102) and McFabZ (His74 and Tyr112) enzymes. Both complexes have shown the structural stability during the 20 ns run of molecular dynamics based simulations. Altogether, the present study suggests that NPQ scaffold can be exploited as a multi-targeted inhibitor of FAS II pathway, and these biochemical and biophysical findings will further help in the development of potent antibacterial agents targeting FAS II pathway.

The inhibitory and binding studies of methyl-sulfone hydroxamate based inhibitors against LpxC from drug resistant Moraxella catarrhalis using biophysical, biochemical and in silico approaches.

Several reported potential compounds against UDP-3-O-(R-3-Hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) have shown large variation in the potency and efficacy. The differential susceptibility and selective binding of these inhibitors against LpxC are still unexplored. In the present work, we have characterized LpxC from Moraxella catarrhalis (McLpxC) and investigated its binding with potent inhibitors LpxC-2 and LpxC-4 using biochemical, biophysical and in silico approaches. The circular dichroism studies have revealed the changes in the secondary and tertiary structure of McLpxC upon inhibitors binding. The fluorescence quenching mechanism was found to be static with kq > 1010 suggesting the ground state complex formation between the McLpxC and inhibitors. Altogether spectroscopic findings suggest that the interaction of LpxC-4 and LpxC-2 caused conformational changes marked by the loss of α-helical content in McLpxC. In ITC based studies, both inhibitors have shown comparable binding affinities (KD = ~10.0 µΜ), and their interactions were exothermically driven by enthalpy change. The docking studies have shown the possibility of two binding sites in McLpxC for these inhibitors with similar binding energies (~10.0 kcal mol-1). Thus, the present study significantly suggests that further optimization and utilization of molecules based on this scaffold will be helpful in designing the new antimicrobial agents targeting LpxC.

ß-Lactamase production and antibiotic susceptibility pattern of Moraxella catarrhalis isolates collected from two county hospitals in China.

BACKGROUND: Moraxella catarrhalis (M. catarrhalis) is an important bacterial pathogen. However, its antibiotic susceptibility patterns in different areas are difficult to compare because of the use of different methods and judgement criteria. This study aimed to determine antimicrobial susceptibility and ß-lactamase activity characteristics of M. catarrhalis isolates collected from two county hospitals in China, and to express the results with reference to three commonly used judgement criteria. RESULTS: Nasopharyngeal swabs were obtained from child inpatients with respiratory tract infections at the People's Hospital of Zhongjiang County and Youyang County from January to December 2015. M. catarrhalis strains were isolated and identified from the swabs, and susceptibility against 11 antimicrobials was determined using the E-test method or disc diffusion. Test results were interpreted with reference to the standards of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the Clinical and Laboratory Standards Institute (CLSI), and the British Society for Antimicrobial Chemotherapy (BSAC). Detection of ß-lactamase activity was determined by the chromogenic cephalosporin nitrocefin. M. catarrhalis yield rates were 7.12 and 9.58% (Zhongjiang County, 77/1082 cases; Youyang County, 101/1054 cases, respectively). All isolates were susceptible to amoxicillin-clavulanic acid. The susceptibility rate to meropenem was 100% according to EUCAST; no breakpoints were listed in CLSI or BSAC. The non-susceptibility rate to sulfamethoxazole-trimethoprim differed significantly between the two hospitals regardless of the judgemnet criteria used, with isolates from Zhongjiang showing higher susceptibility to those from Youyang (Fisher's exact test, P < 0.05). According to CLSI, the total non-susceptibility rate to erythromycin was 70.8% (Zhongjiang County, 79.2%; Youyang County, 64.3%), and the rate reached 92.1% (Zhongjiang County, 90.9%; Youyang County, 93.1%) on the basis of EUCAST or BSAC. The total positive rate of ß-lactamase was 99.4% (177/178 cases) (Zhongjiang County, 100%, 77/77 cases; Youyang County, 99.0%, 100/101 cases). CONCLUSIONS: Ninety nine percent of M. catarrhalis isolates produce ß-lactamase. The isolates showed poor susceptibility to ampicillin and erythromycin, and high susceptibility to the third- and fourth-generation cephalosporins and amoxicillin-clavulanic. Significant discrepancies between different antimicrobial susceptibility judgemnet criteria were noted.

Substrate Recognition by a Colistin Resistance Enzyme from Moraxella catarrhalis.

Lipid A phosphoethanolamine (PEtN) transferases render bacteria resistant to the last resort antibiotic colistin. The recent discoveries of pathogenic bacteria harboring plasmid-borne PEtN transferase ( mcr) genes have illustrated the serious potential for wide dissemination of these resistance elements. The origin of mcr-1 is traced to Moraxella species co-occupying environmental niches with Enterobacteriaceae. Here, we describe the crystal structure of the catalytic domain of the chromosomally encoded colistin resistance PEtN transferase, ICR Mc (for intrinsic colistin resistance) of Moraxella catarrhalis. The ICR Mc structure in complex with PEtN reveals key molecular details including specific residues involved in catalysis and PEtN binding. It also demonstrates that ICR Mc catalytic domain dimerization is required for substrate binding. Our structure-guided phylogenetic analysis provides sequence signatures defining potentially colistin-active representatives in this enzyme family. Combined, these results advance the molecular and mechanistic understanding of PEtN transferases and illuminate their origins.

In Vitro and In Vivo Activities of DS-2969b, a Novel GyrB Inhibitor, and Its Water-Soluble Prodrug, DS11960558, against Methicillin-Resistant Staphylococcus aureus.

DS-2969b is a novel GyrB inhibitor under clinical development. In this study, the in vitro activity of DS-2969b and the in vivo activities of DS-2969b and its water-soluble prodrug, DS11960558, against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. DS-2969b inhibited the supercoiling activity of S. aureus DNA gyrase and the decatenation activity of its topoisomerase IV. DS-2969b showed antibacterial activity against Gram-positive aerobes but not against Gram-negative aerobes, except for Moraxella catarrhalis and Haemophilus influenzae DS-2969b was active against MRSA with an MIC90 of 0.25 µg/ml, which was 8-fold lower than that of linezolid. The presence of a pulmonary surfactant did not affect the MIC of DS-2969b. DS-2969b showed time-dependent slow killing against MRSA. The frequency of spontaneous resistance development was less than 6.2 × 10-10 in all four S. aureus isolates at 4× MIC of DS-2969b. In a neutropenic MRSA-induced murine muscle infection model, DS-2969b was more efficacious than linezolid by both the subcutaneous and oral routes. DS-2969b and DS11960558 showed efficacy in a neutropenic murine MRSA lung infection model. The pharmacokinetics and pharmacodynamics of DS-2969b and DS11960558 against MRSA were characterized in a neutropenic murine thigh infection model; the percentage of time during the dosing period in which the free drug concentration exceeded the MIC (fTMIC) correlated best with in vivo efficacy, and the static percent fTMIC was 43 to 49%. A sufficient fTMIC was observed in a phase 1 multiple-ascending-dose study of DS-2969b given orally at 400 mg once a day. These results suggest that DS11960558 and DS-2969b have potential for use as intravenous-to-oral step-down therapy for treating MRSA infections with a higher efficacy than linezolid.

Mapping Protective Regions on a Three-Dimensional Model of the Moraxella catarrhalis Vaccine Antigen Oligopeptide Permease A.

A vaccine against Moraxella catarrhalis would reduce tremendous morbidity, mortality, and financial burden by preventing otitis media in children and exacerbations of chronic obstructive pulmonary disease (COPD) in adults. Oligopeptide permease A (OppA) is a candidate vaccine antigen that is (i) a nutritional virulence factor expressed on the bacterial cell surface during infection, (ii) widely conserved among strains, (iii) highly immunogenic, and (iv) a protective antigen based on its capacity to induce protective responses in immunized animals. In the present study, we show that the antibodies to OppA following vaccination mediate accelerated clearance in animals after pulmonary challenge. To identify regions of OppA that bind protective antibodies, truncated constructs of OppA were engineered and studied to map regions of OppA with surface-accessible epitopes that bind high-avidity antibodies following vaccination. Protective epitopes were located in the N and C termini of the protein. Immunization of mice with constructs corresponding to these regions (T5 and T8) induced protective responses. Studies of overlapping peptide libraries of constructs T5 and T8 with OppA immune serum identified two discrete regions on each construct. These potentially protective regions were mapped on a three-dimensional computational model of OppA, where regions with solvent-accessible amino acids were identified as three potentially protective epitopes. In all, these studies revealed two regions with three specific epitopes in OppA that induce potentially protective antibody responses following vaccination. Detection of antibodies to these regions could serve to guide vaccine formulation and as a diagnostic tool for monitoring development of protective responses during clinical trials.

Characterization of isoflavonoids as inhibitors of ß-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Moraxella catarrhalis: Kinetics, spectroscopic, thermodynamics and in silico studies.

BACKGROUD: ß-hydroxyacyl-acyl carrier protein dehydratase (FabZ) is an essential component of type II fatty acid biosynthesis (FAS II) pathway in bacteria. It performs dehydration of ß-hydroxyacyl-ACP to trans-2-acyl-ACP in the elongation cycle of the FAS II pathway. FabZ is ubiquitously expressed and has uniform distribution, which makes FabZ an excellent target for developing novel drugs against pathogenic bacteria. METHODS: We focused on the biochemical and biophysical characterization of FabZ from drug-resistant pathogen Moraxella catarrhalis (McFabZ). More importantly, we have identified and characterized new inhibitors against McFabZ using biochemical, biophysical and in silico based studies. RESULTS: We have identified three isoflavones (daidzein, biochanin A and genistein) as novel inhibitors against McFabZ. Mode of inhibition of these compounds is competitive with IC50 values lie in the range of 6.85µΜ to 27.7µΜ. Conformational changes observed in secondary and tertiary structure marked by a decrease in the helical and the sheet content in McFabZ structure upon inhibitors binding. In addition, thermodynamic data suggest that biochanin A has a strong binding affinity for McFabZ as compare to daidzein and genistein. Molecular docking studies have revealed that these inhibitors are interacting with the active site of McFabZ and making contacts with catalytic and substrate binding tunnel residues. CONCLUSION AND GENERAL SIGNIFICANCE: Three new inhibitors against McFabZ have been identified and characterized. These biochemical and biophysical findings lead to the identification of chemical scaffolds, which can lead to broad-spectrum antimicrobial drugs targeted against FabZ, and modification to existing FabZ inhibitors to improve affinity and potency.

Acyl chain preference and inhibitor identification of Moraxella catarrhalis LpxA: Insight through crystal structure and computational studies.

Lipopolysaccharide (LPS) is an important surface component and a potential virulence factor in the pathogenesis of Gram-negative bacteria. UDP-N-acetylglucosamine acyltransferase (LpxA) enzyme catalyzes the first reaction of LPS biosynthesis, reversible transfer of R-3-hydroxy-acyl moiety from donor R-3-hydroxy-acyl-acyl carrier protein to the 3' hydroxyl position of UDP-N-acetyl-glucosamine. LpxA enzyme's essentiality in bacterial survival and absence of any homologous protein in humans makes it a promising target for anti-bacterial drug development. Herein, we present the crystal structure of Moraxella catarrhalis LpxA (McLpxA). We propose that L171 is responsible for limiting the acyl chain length in McLpxA to 10C or 12C. The study reveals the plausible interactions between the highly conserved clusters of basic residues at the C-terminal end of McLpxA and acidic residues of acyl carrier protein (ACP). Furthermore, the crystal structure of McLpxA was used to screen potential inhibitors from NCI open database using various computational approaches viz. pharmacophore mapping, virtual screening and molecular docking. Molecules Mol212032, Mol609399 and Mol152546 showed best binding affinity with McLpxA among all screened molecules. These molecules mimic the substrate-LpxA binding interactions.

Antimicrobial susceptibility to ß-lactam antibiotics and production of BRO ß-lactamase in clinical isolates of Moraxella catarrhalis from a Japanese hospital.

We investigated BRO-ß-lactamase production of Moraxella catarrhalis isolates and its antimicrobial susceptibility to ß-lactams. Of the 233 isolates, 232 were BRO producers and 224 were BRO-1 producers. Four isolates exhibited elevated ceftriaxone minimum inhibitory concentration (2 µg/mL) and different pulsed-field gel electrophoresis patterns and we expect this number to increase in the near future.

Moraxella catarrhalis-produced nitric oxide has dual roles in pathogenicity and clearance of infection in bacterial-host cell co-cultures.

In humans, the free radical nitric oxide (NO) is a concentration-dependent multifunctional signaling or toxic molecule that modulates various physiological and pathological processes, and innate immunity against bacterial infections. Because the expression of bacterial genes encoding nitrite reductase (AniA) and NO reductase (NorB) is highly upregulated in biofilms in vitro, it is important to investigate whether bacterial NO-metabolism might subvert host NO signaling and play pathogenic roles during infection. The Moraxella catarrhalis AniA and NorB directly function in production and reduction of NO. Using M. catarrhalis-human bronchial epithelial cell (HBEC) co-cultures, we recently reported AniA/nitrite-dependent cytotoxic effects on HBECs, including altered protein profiles of HBECs and induced HBEC apoptosis, suggesting bacterial nitrite reduction likely dysregulates host cell gene expression. To further clarify whether nitrite reduction-derived NO or nitrite-dependent stimulation of bacterial growth was responsible for adverse effects on HBECs, we monitored bacterial nitrite reduction, levels of NO in co-cultures and resulted dynamic effects on HBEC proliferation and bacterial viability. This study demonstrated that M. catarrhalis nitrite reduction-derived NO was responsible for observed adverse effects on HBECs at mid-to-late stages of infection. More importantly, our data showed that while nitrite promoted bacterial growth and biofilm formation at early hours of infection, nitrite reduction-derived NO was toxic towards M. catarrhalis in maturing biofilms, suggesting nitrite reduction-derived NO might be a possible dualistic mechanism by which M. catarrhalis promotes diseases and spontaneous resolutions.

Expression of the Oligopeptide Permease Operon of Moraxella catarrhalis Is Regulated by Temperature and Nutrient Availability.

Moraxella catarrhalis causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. Together, these two conditions contribute to enormous morbidity and mortality worldwide. The oligopeptide permease (opp) ABC transport system is a nutritional virulence factor important for the utilization of peptides. The substrate binding protein OppA, which binds peptides for uptake, is a potential vaccine antigen, but little was known about the regulation of gene expression. The five opp genes oppB, oppC, oppD, oppF, and oppA are in the same open reading frame. Sequence analysis predicted two promoters, one located upstream of oppB and one within the intergenic region between oppF and oppA. We have characterized the gene cluster as an operon with two functional promoters and show that cold shock at 26°C for ≤ 0.5 h and the presence of a peptide substrate increase gene transcript levels. Additionally, the putative promoter upstream of oppA contributes to the transcription of oppA but is not influenced by the same environmental cues as the promoter upstream of oppB. We conclude that temperature and nutrient availability contribute to the regulation of the Opp system, which is an important nutritional virulence factor in M. catarrhalis.

Ligand-bound structures of 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Moraxella catarrhalis reveal a water channel connecting to the active site for the second step of catalysis.

KdsC, the third enzyme of the 3-deoxy-D-manno-octulosonic acid (KDO) biosynthetic pathway, catalyzes a substrate-specific reaction to hydrolyze 3-deoxy-D-manno-octulosonate 8-phosphate to generate a molecule of KDO and phosphate. KdsC is a phosphatase that belongs to the C0 subfamily of the HAD superfamily. To understand the molecular basis for the substrate specificity of this tetrameric enzyme, the crystal structures of KdsC from Moraxella catarrhalis (Mc-KdsC) with several combinations of ligands, namely metal ion, citrate and products, were determined. Various transition states of the enzyme have been captured in these crystal forms. The ligand-free and ligand-bound crystal forms reveal that the binding of ligands does not cause any specific conformational changes in the active site. However, the electron-density maps clearly showed that the conformation of KDO as a substrate is different from the conformation adopted by KDO when it binds as a cleaved product. Furthermore, structural evidence for the existence of an intersubunit tunnel has been reported for the first time in the C0 subfamily of enzymes. A role for this tunnel in transferring water molecules from the interior of the tetrameric structure to the active-site cleft has been proposed. At the active site, water molecules are required for the formation of a water bridge that participates as a proton shuttle during the second step of the two-step phosphoryl-transfer reaction. In addition, as the KDO biosynthesis pathway is a potential antibacterial target, pharmacophore-based virtual screening was employed to identify inhibitor molecules for the Mc-KdsC enzyme.

Non-transpeptidase binding arylthioether ß-lactams active against Mycobacterium tuberculosis and Moraxella catarrhalis.

The prevalence of drug resistance in both clinical and community settings as a consequence of alterations of biosynthetic pathways, enzymes or cell wall architecture is a persistent threat to human health. We have designed, synthesized, and tested a novel class of non-transpeptidase, ß-lactamase resistant monocyclic ß-lactams that carry an arylthio group at C4. These thioethers exhibit inhibitory and cidal activity against serine ß-lactamase producing Mycobacterium tuberculosis wild type strain (Mtb) and multiple (n=8) ß-lactamase producing Moraxella catarrhalis clinical isolates.

Role of the oligopeptide permease ABC Transporter of Moraxella catarrhalis in nutrient acquisition and persistence in the respiratory tract.

Moraxella catarrhalis is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality. M. catarrhalis has a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival. M. catarrhalis has a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB, oppC, oppD, oppF, and oppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the oppBCDFA gene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth of M. catarrhalis is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-type M. catarrhalis was observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requires l-arginine. An oppA knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract.

The frequency of BRO ß-lactamase and its relationship to antimicrobial susceptibility and serum resistance in Moraxella catarrhalis.

We investigated the frequency of BRO ß-lactamase and its relationship to antibiotic susceptibility profiles and serum susceptibility. Moraxella catarrhalis clinical isolates (n = 197) were collected from patients with respiratory tract infections in Tokyo between November 2004 and April 2005. Phenotypic and genotypic detection of ß-lactamases was performed. The MICs of 6 antibiotics were determined by Etest, and the serum bactericidal assay was conducted by using the culture-and-spot test. Nearly all (192; 97%) of the clinical isolates were ß-lactamase producers; of these, 182 (95%) were bro-1 and 10 (5%) were bro-2 positive. MIC50, MIC90, and geometric mean MICs of penicillin, amoxicillin, cefixime, and clarithromycin for BRO-1 isolates were significantly higher than for BRO-2 isolates. The frequency of intermediate and full serum resistance was significantly higher in BRO-1 isolates than in BRO-2 isolates (P = 0.0056), but not BRO-negative isolates (P = 0.1333). We provide the first evidence that the presence of BRO-1 in M. catarrhalis is associated with reduced susceptibility to clarithromycin and ß-lactam antibiotics, as well as serum non-sensitive (intermediate and resistant).

Deciphering the genetic basis of Moraxella catarrhalis complement resistance: a critical role for the disulphide bond formation system.

The complement system is an important innate defence mechanism, and the ability to resist complement-mediated killing is considered a key virulence trait of the respiratory tract pathogen M. catarrhalis. We studied the molecular basis of complement resistance by transcriptional profiling and Tn-seq, a genome-wide negative-selection screenings technology. Exposure of M. catarrhalis to human serum resulted in increased expression of 84 genes and reduced expression of 134 genes, among which genes encoding ABC transporter systems and surface proteins UspA1 and McaP. By subjecting a ∼ 15 800 transposon mutant library to serum, mutants of 53 genes were negatively selected, including the key complement-resistance factor uspA2H. Validation with directed mutants confirmed Tn-seq phenotypes of uspA2H and 11 newly identified genes, with mutants of MCR_0424, olpA, MCR_1483, and dsbB most severely attenuated. Detailed analysis showed that both components of the disulphide bond formation (DSB) system, DsbB and DsbA, were required for complement-resistance in multiple isolates, and fulfil a critical role in evasion of IgG-dependent classical pathway-mediated killing. Lipooligosaccharide (LOS) structure and membrane stability were severely affected in ΔdsbA strains, suggesting a pivotal role for the DSB system in LOS structure safeguarding and membrane stability maintenance.

Moraxella catarrhalis expresses a cardiolipin synthase that impacts adherence to human epithelial cells.

The major phospholipid constituents of Moraxella catarrhalis membranes are phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin (CL). However, very little is known regarding the synthesis and function of these phospholipids in M. catarrhalis. In this study, we discovered that M. catarrhalis expresses a cardiolipin synthase (CLS), termed MclS, that is responsible for the synthesis of CL within the bacterium. The nucleotide sequence of mclS is highly conserved among M. catarrhalis isolates and is predicted to encode a protein with significant amino acid similarity to the recently characterized YmdC/ClsC protein of Escherichia coli. Isogenic mclS mutant strains were generated in M. catarrhalis isolates O35E, O12E, and McGHS1 and contained no observable levels of CL. Site-directed mutagenesis of a highly conserved HKD motif of MclS also resulted in a CL-deficient strain. Moraxella catarrhalis, which depends on adherence to epithelial cells for colonization of the human host, displays significantly reduced levels of adherence to HEp-2 and A549 cell lines in the mclS mutant strains compared to wild-type bacteria. The reduction in adherence appears to be attributed to the absence of CL. These findings mark the first instance in which a CLS has been related to a virulence-associated trait.