Evolution of bone compactness in extant and extinct moles (Talpidae): exploring humeral microstructure in small fossorial mammals.

BACKGROUND: Talpids include forms with different degree of fossoriality, with major specializations in the humerus in the case of the fully fossorial moles. We studied the humeral microanatomy of eleven extant and eight extinct talpid taxa of different lifestyles and of two non-fossorial outgroups and examined the effects of size and phylogeny. We tested the hypothesis that bone microanatomy is different in highly derived humeri of fossorial taxa than in terrestrial and semi-aquatic ones, likely due to special mechanical strains to which they are exposed to during digging. This study is the first comprehensive examination of histological parameters in an ecologically diverse and small-sized mammalian clade. RESULTS: No pattern of global bone compactness was found in the humeri of talpids that could be related to biomechanical specialization, phylogeny or size. The transition zone from the medullary cavity to the cortical compacta was larger and the ellipse ratio smaller in fossorial talpids than in non-fossorial talpids. No differences were detected between the two distantly related fossorial clades, Talpini and Scalopini. CONCLUSIONS: At this small size, the overall morphology of the humerus plays a predominant role in absorbing the load, and microanatomical features such as an increase in bone compactness are less important, perhaps due to insufficient gravitational effects. The ellipse ratio of bone compactness shows relatively high intraspecific variation, and therefore predictions from this ratio based on single specimens are invalid.

Induction kinetics of the Staphylococcus aureus cell wall stress stimulon in response to different cell wall active antibiotics.

BACKGROUND: Staphylococcus aureus activates a protective cell wall stress stimulon (CWSS) in response to the inhibition of cell wall synthesis or cell envelope damage caused by several structurally and functionally different antibiotics. CWSS induction is coordinated by the VraSR two-component system, which senses an unknown signal triggered by diverse cell wall active agents. RESULTS: We have constructed a highly sensitive luciferase reporter gene system, using the promoter of sas016 (S. aureus N315), which detects very subtle differences in expression as well as measuring > 4 log-fold changes in CWSS activity, to compare the concentration dependence of CWSS induction kinetics of antibiotics with different cell envelope targets. We compared the effects of subinhibitory up to suprainhibitory concentrations of fosfomycin, D-cycloserine, tunicamycin, bacitracin, flavomycin, vancomycin, teicoplanin, oxacillin, lysostaphin and daptomycin. Induction kinetics were both strongly antibiotic- and concentration-dependent. Most antibiotics triggered an immediate response with induction beginning within 10 min, except for tunicamycin, D-cycloserine and fosfomycin which showed lags of up to one generation before induction commenced. Induction characteristics, such as the rate of CWSS induction once initiated and maximal induction reached, were strongly antibiotic dependent. We observed a clear correlation between the inhibitory effects of specific antibiotic concentrations on growth and corresponding increases in CWSS induction kinetics. Inactivation of VraR increased susceptibility to the antibiotics tested from 2- to 16-fold, with the exceptions of oxacillin and D-cycloserine, where no differences were detected in the methicillin susceptible S. aureus strain background analysed. There was no apparent correlation between the induction capacity of the various antibiotics and the relative importance of the CWSS for the corresponding resistance phenotypes. CONCLUSION: CWSS induction profiles were unique for each antibiotic. Differences observed in optimal induction conditions for specific antibiotics should be determined and taken into account when designing and interpreting CWSS induction studies.

Phylogenetic distribution and membrane topology of the LytR-CpsA-Psr protein family.

BACKGROUND: The bacterial cell wall is the target of many antibiotics and cell envelope constituents are critical to host-pathogen interactions. To combat resistance development and virulence, a detailed knowledge of the individual factors involved is essential. Members of the LytR-CpsA-Psr family of cell envelope-associated attenuators are relevant for beta-lactam resistance, biofilm formation, and stress tolerance, and they are suggested to play a role in cell wall maintenance. However, their precise function is still unknown. This study addresses the occurrence as well as sequence-based characteristics of the LytR-CpsA-Psr proteins. RESULTS: A comprehensive list of LytR-CpsA-Psr proteins was established, and their phylogenetic distribution and clustering into subgroups was determined. LytR-CpsA-Psr proteins were present in all Gram-positive organisms, except for the cell wall-deficient Mollicutes and one strain of the Clostridiales. In contrast, the majority of Gram-negatives did not contain LytR-CpsA-Psr family members. Despite high sequence divergence, the LytR-CpsA-Psr domains of different subclusters shared a highly similar, predicted mixed a/beta-structure, and conserved charged residues. PhoA fusion experiments, using MsrR of Staphylococcus aureus, confirmed membrane topology predictions and extracellular location of its LytR-CpsA-Psr domain. CONCLUSION: The LytR-CpsA-Psr domain is unique to bacteria. The presence of diverse subgroups within the LytR-CpsA-Psr family might indicate functional differences, and could explain variations in phenotypes of respective mutants reported. The identified conserved structural elements and amino acids are likely to be important for the function of the domain and will help to guide future studies of the LytR-CpsA-Psr proteins.

Role of endogenous Fas (CD95/Apo-1) ligand in balloon-induced apoptosis, inflammation, and neointima formation.

BACKGROUND: Fas (CD95/Apo-1) ligand (FasL)-induced apoptosis in Fas-bearing cells is critically involved in modulating immune reactions and tissue repair. Apoptosis has also been described after mechanical vascular injury such as percutaneous coronary intervention. However, the relevance of cell death in this context of vascular repair remains unknown. METHODS AND RESULTS: To determine whether FasL-induced apoptosis is causally related to neointimal lesion formation, we subjected FasL-deficient (generalized lymphoproliferative disorder [gld], C57BL/6J) and corresponding wild-type (WT) mice to carotid balloon distension injury, which induces marked endothelial denudation and medial cell death. FasL expression in WT mice was induced in injured vessels compared with untreated arteries (P<0.05; n=5). Conversely, absence of functional FasL in gld mice decreased medial and intimal apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling [TUNEL] index) at 1 hour and 7 days after balloon injury (P<0.05; n=6). In addition, peritoneal macrophages isolated from gld mice showed no apoptosis and enhanced migration (P<0.05; n=4). In parallel, we observed increased balloon-induced macrophage infiltrations (anti-CD68) in injured arteries of FasL-deficient animals (P<0.05; n=6). Together with enhanced proliferation (bromodeoxyuridine index; P<0.05), these events resulted in a further increase in medial and neointimal cells (P<0.01; n=8) with thickened neointima in gld mice (intima/media ratio, x3.8 of WT; P<0.01). CONCLUSIONS: Our data identify proapoptotic and antiinflammatory effects of endogenous FasL as important factors in the process of neointimal lesion formation after balloon injury. Moreover, they suggest that activation of FasL may decrease neointimal thickening after percutaneous coronary intervention.

18F-choline images murine atherosclerotic plaques ex vivo.

OBJECTIVE: Current imaging modalities of atherosclerosis mainly visualize plaque morphology. Valuable insight into plaque biology was achieved by visualizing enhanced metabolism in plaque-derived macrophages using 18F-fluorodeoxyglucose (18F-FDG). Similarly, enhanced uptake of 18F-fluorocholine (18F-FCH) was associated with macrophages surrounding an abscess. As macrophages are important determinants of plaque vulnerability, we tested 18F-FCH for plaque imaging. METHODS AND RESULTS: We injected 18F-FCH (n=5) or 18F-FDG (n=5) intravenously into atherosclerotic apolipoprotein E-deficient mice. En face measurements of aortae isolated 20 minutes after 18F-FCH injections demonstrated an excellent correlation between fat stainings and autoradiographies (r=0.842, P<0.0001), achieving a sensitivity of 84% to detect plaques by 18F-FCH. In contrast, radiotracer uptake 20 minutes after 18F-FDG injections correlated less with en face fat stainings (r=0.261, P<0.05), reaching a sensitivity of 64%. Histological analyses of cross-sections 20 minutes after coinjections of 18F-FCH and 14C-FDG (n=3) showed that 18F-FCH uptake correlated better with fat staining (r=0.740, P<0.0001) and macrophage-positive areas (r=0.740, P<0.0001) than 14C-FDG (fat: r=0.236, P=0.29 and CD68 staining: r=0.352, P=0.11), respectively. CONCLUSIONS: 18F-FCH identifies murine plaques better than 18F-FDG using ex vivo imaging. Enhanced 18F-FCH uptake into macrophages may render this tracer a promising candidate for imaging plaques in patients.

Increased balloon-induced inflammation, proliferation, and neointima formation in apolipoprotein E (ApoE) knockout mice.

BACKGROUND AND PURPOSE: The pathophysiology of vascular lesions after balloon angioplasty remains poorly understood. A major limitation of most experimental studies in this regard is that injury was assessed in healthy arteries. Our aim was to study the effects of hypercholesterolemia in a mouse vascular injury model that mimics human balloon angioplasty. METHODS: Carotid balloon distension was performed in wild-type (WT) mice on a normal diet (ND), in apolipoprotein E-deficient (ApoE-/-) mice on ND and in ApoE-/- mice fed a high cholesterol diet (CD). RESULTS: Medial cell death (TUNEL) was elevated in all mice at 1 hour and 1 day after angioplasty without differences between the groups. We found enhanced intimal inflammation (%CD45-positive cells) and vascular cell adhesion molecule-1 expression at 7 days (P < 0.05; n > or = 4) as well as increased proliferation rates (BrdU-index) in ApoE-/- CD at 7 and 28 days postinjury (P < 0.05; n > or = 5). Four weeks after injury, these events led to enhanced neointima in ApoE-/- CD compared with WT ND mice (intima/media, P < 0.001; n > or = 8). The amount of lesion formation paralleled the incremental increase in total plasma cholesterol in WT ND, ApoE-/- ND and ApoE-/- CD (P < 0.01). CONCLUSIONS: Carotid balloon distension injury in ApoE-/- mice on CD induced enhanced inflammation and proliferation leading to increased neointima. Further applications of this microballoon catheter in genetically modified mice will provide opportunities to elucidate molecular mechanisms of vascular lesion formation in a model that reflects clinical balloon angioplasty. This know-how may pave the way to catheter-based interventions of human microvessels in the peripheral or cerebral circulation.

Unveiling electrotransformation of Moraxella catarrhalis as a process of natural transformation.

The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhalis.

Molecular imaging of atherosclerotic plaques using a human antibody against the extra-domain B of fibronectin.

Current imaging modalities of human atherosclerosis, such as angiography, ultrasound, and computed tomography, visualize plaque morphology. However, methods that provide insight into plaque biology using molecular tools are still insufficient. The extra-domain B (ED-B) is inserted into the fibronectin molecule by alternative splicing during angiogenesis and tissue remodeling but is virtually undetectable in normal adult tissues. Angiogenesis and tissue repair are also hallmarks of advanced plaques. For imaging atherosclerotic plaques, the human antibody L19 (specific against ED-B) and a negative control antibody were labeled with radioiodine or infrared fluorophores and injected intravenously into atherosclerotic apolipoprotein E-null (ApoE-/-) or normal wild-type mice. Aortas isolated 4 hours, 24 hours, and 3 days after injection exhibited a selective and stable uptake of L19 when using radiographic or fluorescent imaging. L19 binding was confined to the plaques as assessed by fat staining. Comparisons between fat staining and autoradiographies 24 hours after 125I-labeled L19 revealed a significant correlation (r=0.89; P<0.0001). Minimal antibody uptake was observed in normal vessels from wild-type mice receiving the L19 antibody and in atherosclerotic vessels from ApoE-/- mice receiving the negative control antibody. Immunohistochemical studies revealed increased expression of ED-B not only in murine but also in human plaques, in which it was found predominantly around vasa vasorum and plaque matrix. In summary, we demonstrate selective targeting of atheromas in mice using the human antibody to the ED-B domain of fibronectin. Thus, our findings may set the stage for antibody-based molecular imaging of atherosclerotic plaques in the intact organism.

The modern autopsy: what to do if infection is suspected.

Deaths due to infectious diseases are common worldwide. The autopsy, although less frequently performed than previously, is important to our understanding of disease pathogenesis. The autopsy also provides critical information regarding potential disease outbreaks. To optimize the benefits of an autopsy, the pathologist should approach the autopsy with a well-constructed differential diagnosis that provides the framework for appropriate selection of diagnostic specimens and tests. Standard microbiologic cultures, although necessary and important, are often insufficient and must be supplemented by newer molecular methodologies.

Mucosal immune response to specific outer membrane proteins of Moraxella catarrhalis in young children.

BACKGROUND: Moraxella catarrhalis is an important cause of otitis media. A number of candidate antigens for a future infant otitis media vaccine have been identified, but their mucosal immunogenicity induced by nasopharyngeal M. catarrhalis colonization has not been characterized. The aim of this study was to determine the salivary IgA response to M. catarrhalis outer membrane proteins (OMP) in young children. METHODS: Children ages 1 to 24 months evaluated for acute respiratory tract infection were prospectively enrolled. M. catarrhalis nasopharyngeal colonization was determined by (1) selective culture and (2) detection by reverse transcription-PCR of messenger RNA specific for the OMP UspA1 and UspA2. Salivary IgA responses were detected by immunoblot analysis of M. catarrhalis OMP. Isogenic knockout mutants for UspA1, UspA2, hemagglutinin (Hag), transferrin-binding protein B (TbpB) and CopB were constructed for identification of specific target OMP. RESULTS: Sixty-six patients were studied. The rates of M. catarrhalis colonization by culture, reverse transcription-PCR for messenger RNA and mRNA were 40, 94 and 58%, respectively. Anti-M. catarrhalis salivary IgA was detected in 62 patients (94%). IgA directed against a >250-kDa antigen (assigned to UspA1/UspA2 by mutant analysis) and a 200-kDa antigen (Hag) were detected in 65 and 70% of patients, respectively. Bands at 80 to 85 kDa (82%) consisted of IgA directed against monomeric UspA2, TbpB and CopB. CONCLUSIONS: colonization occurring in early infancy is associated with a consistent mucosal immune response directed against the UspA proteins, Hag and other OMP. The data suggest that several M. catarrhalis OMP are immunogens of the nasopharyngeal mucosal immune system of infants.

The effect of moving to a new hospital facility on the prevalence of methicillin-resistant Staphylococcus aureus.

BACKGROUND: The influence of hospital design on nosocomial transmission of methicillin-resistant Staphylococcus aureus (MRSA) is unknown. Our hospital's relocation to a new building with radically different ward design allowed us to study this question. Our old hospital facility had open bay wards and intensive care units, and few poorly located sinks for handwashing (bed:sink ratio 4:1). Our new hospital facility had optimized hand-washing geography and distribution of ward beds into mostly single or double rooms (bed:sink ratio 1.3:1). METHODS: We compared the prevalence of MRSA in the 2 institutions by obtaining nasal swabs from all patients on 8 selected wards and intensive care units at 2 time points both before and after the move. In addition, passive surveillance rates of MRSA for all hospitalized patients for 2 years both before and after the move were compared. Hand hygiene practices, although unrelated to the study periods, were directly observed. RESULTS: Eight of 123 patients cultured before the move were MRSA positive, compared to 5 of 138 patients cultured after the move (P=NS). MRSA prevalence determined by passive surveillance of all hospitalized patients before and after the move was also unchanged. An insignificant increase in the frequency of hand-hygiene performance following the move (20% to 23%) was observed. CONCLUSION: Radical facility design changes, which would be permissive of optimal infection control practices, were not sufficient, by themselves, to reduce the nosocomial spread of MRSA in our institution.

Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response.

The Staphylococcus aureus cell wall stress stimulon (CWSS) is activated by cell envelope-targeting antibiotics or depletion of essential cell wall biosynthesis enzymes. The functionally uncharacterized S. aureus LytR-CpsA-Psr (LCP) proteins, MsrR, SA0908 and SA2103, all belong to the CWSS. Although not essential, deletion of all three LCP proteins severely impairs cell division. We show here that VraSR-dependent CWSS expression was up to 250-fold higher in single, double and triple LCP mutants than in wild type S. aureus in the absence of external stress. The LCP triple mutant was virtually depleted of wall teichoic acids (WTA), which could be restored to different degrees by any of the single LCP proteins. Subinhibitory concentrations of tunicamycin, which inhibits the first WTA synthesis enzyme TarO (TagO), could partially complement the severe growth defect of the LCP triple mutant. Both of the latter findings support a role for S. aureus LCP proteins in late WTA synthesis, as in Bacillus subtilis where LCP proteins were recently proposed to transfer WTA from lipid carriers to the cell wall peptidoglycan. Intrinsic activation of the CWSS upon LCP deletion and the fact that LCP proteins were essential for WTA-loading of the cell wall, highlight their important role(s) in S. aureus cell envelope biogenesis.

LytR-CpsA-Psr proteins in Staphylococcus aureus display partial functional redundancy and the deletion of all three severely impairs septum placement and cell separation.

Staphylococcus aureus contains three members of the LytR-CpsA-Psr (LCP) family of membrane proteins: MsrR, SA0908 and SA2103. The characterization of single-, double- and triple-deletion mutants revealed distinct phenotypes for each of the three proteins. MsrR was involved in cell separation and septum formation and influenced ß-lactam resistance; SA0908 protected cells from autolysis; and SA2103, although displaying no apparent phenotype by itself, enhanced the properties of msrR and sa0908 mutants when deleted. The deletion of sa0908 and sa2103 also further attenuated the virulence of msrR mutants in a nematode-killing assay. The severely defective growth phenotype of the triple mutant revealed that LytR-CpsA-Psr proteins are essential for optimal cell division in S. aureus. Growth could be rescued to varying degrees by any one of the three proteins, indicating some functional redundancy within members of this protein family. However, differing phenotypic characteristics of all single and double mutants and complemented triple mutants indicated that each protein played a distinct role(s) and contributed differently to phenotypes influencing cell separation, autolysis, cell surface properties and virulence.

MsrR contributes to cell surface characteristics and virulence in Staphylococcus aureus.

MsrR, a factor contributing to methicillin resistance in Staphylococcus aureus, belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins. Deletion of msrR increased cell size and aggregation, and altered envelope properties, leading to a temporary reduction in cell surface hydrophobicity, diminished colony-spreading ability, and an increased susceptibility to Congo red. The reduced phosphorus content of purified cell walls of the msrR mutant suggested a reduction in wall teichoic acids, which may explain some of the observed phenotypes. Microarray analysis of the msrR deletion mutant revealed only minor changes in the global transcriptome, suggesting that MsrR has structural rather than regulatory functions. Importantly, virulence of the msrR mutant was decreased in a nematode-killing assay as well as in rat experimental endocarditis. MsrR is therefore likely to play a role in cell envelope maintenance, cell separation, and pathogenicity of S. aureus.

Endogenous IFN-gamma production is induced and required for protective immunity against pulmonary chlamydial infection in neonatal mice.

Chlamydia trachomatis infection in neonates, not adults, has been associated with the development of chronic respiratory sequelae. Adult chlamydial infections induce Th1-type responses that subsequently clear the infection, whereas the neonatal immune milieu in general has been reported to be biased toward Th2-type responses. We examined the protective immune responses against intranasal Chlamydia muridarum challenge in 1-day-old C57BL/6 and BALB/c mice. Infected C57BL/6 pups displayed earlier chlamydial clearance (day 14) compared with BALB/c pups (day 21). However, challenged C57BL/6 pups exhibited prolonged deficits in body weight gain (days 12-30) compared with BALB/c pups (days 9-12), which correlated with continual pulmonary cellular infiltration. Both strains exhibited a robust Th1-type response, including elevated titers of serum antichlamydial IgG2a and IgG2b, not IgG1, and elevated levels of splenic C. muridarum-specific IFN-gamma, not IL-4, production. Additionally, elevated IFN-gamma, not IL-4 expression, was observed locally in the infected lungs of both mouse strains. The immune responses in C57BL/6 pups were significantly greater compared with BALB/c pups after chlamydial challenge. Importantly, infected mice deficient in IFN-gamma or IFN-gamma receptor demonstrated enhanced chlamydial dissemination, and 100% of animals died by 2 wk postchallenge. Collectively, these results indicate that neonatal pulmonary chlamydial infection induces a robust Th1-type response, with elevated pulmonary IFN-gamma production, and that endogenous IFN-gamma is important in protection against this infection. The enhanced IFN-gamma induction in the immature neonatal lung also may be relevant to the development of respiratory sequelae in adult life.

Intranasal vaccination with a secreted chlamydial protein enhances resolution of genital Chlamydia muridarum infection, protects against oviduct pathology, and is highly dependent upon endogenous gamma interferon production.

There is currently no licensed vaccine against Chlamydia trachomatis, the leading cause of sexually transmitted bacterial disease worldwide. Conventional vaccination attempts using surface-exposed chlamydial antigens have achieved only partial success. We have employed a novel vaccination strategy using a secreted protein, chlamydial protease-like activity factor (CPAF), which has been shown to degrade host major histocompatibility complex transcription factors and keratin-8 and therefore may allow immune evasion and establishment of a productive infection. Intranasal immunization using recombinant CPAF (rCPAF) plus interleukin-12 (IL-12) (rCPAF+IL-12 immunization) was used to assess the protective immunity against genital Chlamydia muridarum infection in BALB/c mice. rCPAF+IL-12 immunization induced robust gamma interferon (IFN-gamma) production and minimal IL-4 production by splenocytes upon in vitro recall with rCPAF. The total and immunoglobulin G2a (IgG2a) anti-rCPAF antibody levels in serum were significantly elevated after rCPAF+IL-12 vaccination, as were the total antibody, IgG2a, and IgA levels in bronchoalveolar lavage and vaginal fluids when the animals were compared to animals that received rCPAF alone. rCPAF+IL-12-vaccinated mice displayed significantly reduced bacterial shedding upon chlamydial challenge and accelerated resolution of infection compared to mock-immunized (phosphate-buffered saline) animals. Moreover, rCPAF+IL-12-immunized animals exhibited protection against pathological consequences of chlamydial infection, including the development of hydrosalpinx and oviduct dilatation. This vaccination regimen also reduced the development of fibrosis and the influx of neutrophils into the upper genital tract when the animals were compared to mock-immunized (phosphate-buffered saline) animals after bacterial challenge. rCPAF+IL-12-mediated resolution of the bacterial infection and protection against Chlamydia-induced inflammatory disease were highly dependent on endogenous IFN-gamma production. Together, these results demonstrate that secreted chlamydial antigens may be novel vaccine candidates to induce protective immunity.

The protective efficacy of chlamydial protease-like activity factor vaccination is dependent upon CD4+ T cells.

We have previously determined the protective efficacy of intranasal vaccination with chlamydial protease-like activity factor (CPAF) against genital chlamydial infection. Since T-helper 1 (Th1) responses are important for anti-chlamydial immunity, we examined the contribution of CD4(+) T cells in CPAF mediated immunity against intravaginal (i.vag.) Chlamydia muridarum infection in C57BL/6 mice. CPAF+IL-12 vaccination induced antigen-specific CD4(+) T cells that secreted elevated levels of IFN-gamma, and generated strong humoral responses. The protective effects of CPAF vaccination against genital chlamydial challenge were abrogated by anti-CD4 neutralizing antibody treatment. Moreover, anti-chlamydial immunity could be adoptively transferred to naïve recipients using CPAF-specific CD4(+) T cells. Therefore, CPAF mediated anti-chlamydial immunity is highly dependent upon antigen-specific CD4(+) T cells.

Rapid typing of Moraxella catarrhalis subpopulations based on outer membrane proteins using mass spectrometry.

Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract both in children and in adults. Two subpopulations of this organism have been described that differ in 16S rRNA gene sequence and virulence traits. Three 16S rRNA types have been defined. 2-DE followed by protein identification by MS revealed significant differences in the outer membrane protein (OMP) patterns of each M. catarrhalis 16S rRNA type. Approximately 130 features were detected on the 2-DE map of each M. catarrhalis 16S rRNA type. However, only 50 features were expressed by all strains. Furthermore, direct profiling of isolated OMP using MALDI-TOF MS resulted in a characteristic spectral fingerprint for each 16S rRNA type. Fingerprints remained identical when intact cells instead of isolated OMP were analyzed. This finding suggests that the source of desorbed ions is the outer membrane. Based on the fingerprint we were able to assign 18 well-characterized clinical M. catarrhalis isolates to the correct subpopulation. Therefore, MALDI-TOF of intact M. catarrhalis provides a rapid and robust tool for M. catarrhalis strain typing that could be applied in epidemiological studies.

Cold shock response of the UspA1 outer membrane adhesin of Moraxella catarrhalis.

Colonization of the human nasopharynx exposes Moraxella catarrhalis, a common cause of otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, to sudden downshifts in temperature, occurring when the host breathes cold air. We investigated whether in vitro cold shock influences the expressions of the outer membrane adhesins UspA1 and hemagglutinin, which are considered virulence factors, and of an M. catarrhalis homolog of recA, a housekeeping gene, which in Escherichia coli is induced by cold shock. Quantitative real-time reverse transcriptase PCR was used for measuring mRNA copy number. A screening experiment revealed that a cold shock at 26 degrees C maximally induced the copy number of uspA1. In comparison with 37 degrees C conditions, a 1-hour cold shock at 26 degrees C increased copy numbers of uspA1 and recA by 2.5-fold (11.2 +/- 1.8 versus 4.5 +/- 0.8 copies/CFU) and 2.7-fold (0.30 +/- 0.10 versus 0.11 +/- 0.06), respectively, but did not induce transcription of hag. Exposure to 26 degrees C increased surface expression of UspA1, as assessed by fluorescence-activated cell sorter analysis, and resulted in a significant increase in adherence of strain O35E to Chang human conjunctival cells (97.1% +/- 2.0% versus 48.3% +/- 9.2% at 37 degrees C; P = 0.01). Cold shock induction of uspA1 and recA was detected in strains belonging to either phylogenetic subpopulation of M. catarrhalis (16S rRNA types 1 and 2/3) and was most pronounced in type 2/3 strains (4- to 25-fold for uspA1), which do not express detectable amounts of UspA1 protein at 37 degrees C. These data indicate that cold shock at a physiologically relevant temperature of 26 degrees C induces the expression of at least one virulence factor (UspA1). To our knowledge, no similar data are available for other nasopharyngeal pathogens.

Moraxella catarrhalis strains with reduced expression of the UspA outer membrane proteins belong to a distinct subpopulation.

The outer membrane proteins UspA1 and UspA2 are candidate antigens for a Moraxella catarrhalis vaccine. We previously reported that 103 of 108 isolates (95%) from young children expressed UspA1 detected by reactivity with the monoclonal antibody mAb24B5. The aim of the present study was to investigate mechanisms controlling UspA1 expression by analysis of five mAb24B5 non-reactive isolates. Four of these strains were characterized by (i) decreased or absent transcription of uspA1 and uspA2 and (ii) clustered mutations and deletions in the promoter region of both uspA1 and uspA2. Antigenic or phase variation were not responsible for reduced levels of UspA1 expression. While mAb24B5-positive isolates expressing normal levels of uspA1 and uspA2 mRNA belonged to the previously described 16S rRNA type 1 phylogenetic group, these four mAb24B5-negative isolates were found to belong to the 16S rRNA gene types 2 or 3. The remaining mAb24B5-negative isolate (#610) belonged to 16S rRNA type 1 and exhibited a posttranscriptional defect of UspA1 expression defined by normal levels of uspA1 mRNA and both recombinant and in vitro expression of mAb24B5-reactive UspA1. In conclusion, M. catarrhalis clinical isolates exhibiting reduced expression of UspA1 and UspA2 belonged to a distinct phylogenetic subpopulation. A UspA-based vaccine is unlikely to be effective against such isolates.