Christine Keller

Mycobacterium tuberculosis prevents inflammasome activation.

Mycobacterium tuberculosis (Mtb) parasitizes host macrophages and subverts host innate and adaptive immunity. Several cytokines elicited by Mtb are mediators of mycobacterial clearance or are involved in tuberculosis pathology. Surprisingly, interleukin-1beta (IL-1beta), a major proinflammatory cytokine, has not been implicated in host-Mtb interactions. IL-1beta is activated by processing upon assembly of the inflammasome, a specialized inflammatory caspase-activating protein complex. Here, we show that Mtb prevents inflammasome activation and IL-1beta processing. An Mtb gene, zmp1, which encodes a putative Zn(2+) metalloprotease, is required for this process. Infection of macrophages with zmp1-deleted Mtb triggered activation of the inflammasome, resulting in increased IL-1beta secretion, enhanced maturation of Mtb containing phagosomes, improved mycobacterial clearance by macrophages, and lower bacterial burden in the lungs of aerosol-infected mice. Thus, we uncovered a previously masked role for IL-1beta in the control of Mtb and a mycobacterial system that prevents inflammasome and, therefore, IL-1beta activation.

Prospective randomized controlled multi-centre trial of cuffed or uncuffed endotracheal tubes in small children

BACKGROUNDnThe use of cuffed tracheal tubes (TTs) in small children is still controversial. The aim of this study was to compare post-extubation morbidity and TT exchange rates when using cuffed vs uncuffed tubes in small children.nnnMETHODSnPatients aged from birth to 5 yr requiring general anaesthesia with TT intubation were included in 24 European paediatric anaesthesia centres. Patients were prospectively randomized into a cuffed TT group (Microcuff PET) and an uncuffed TT group (Mallinckrodt, Portex, Rüsch, Sheridan). Endpoints were incidence of post-extubation stridor and the number of TT exchanges to find an appropriate-sized tube. For cuffed TTs, minimal cuff pressure required to seal the airway was noted; maximal cuff pressure was limited at 20 cm H(2)O with a pressure release valve. Data are mean (SD).nnnRESULTSnA total of 2246 children were studied (1119/1127 cuffed/uncuffed). The age was 1.93 (1.48) yr in the cuffed and 1.87 (1.45) yr in the uncuffed groups. Post-extubation stridor was noted in 4.4% of patients with cuffed and in 4.7% with uncuffed TTs (P=0.543). TT exchange rate was 2.1% in the cuffed and 30.8% in the uncuffed groups (P<0.0001). Minimal cuff pressure required to seal the trachea was 10.6 (4.3) cm H(2)O.nnnCONCLUSIONSnThe use of cuffed TTs in small children provides a reliably sealed airway at cuff pressures of <or=20 cm H(2)O, reduces the need for TT exchanges, and does not increase the risk for post-extubation stridor compared with uncuffed TTs.

Genetically Determined Susceptibility to Tuberculosis in Mice Causally Involves Accelerated and Enhanced Recruitment of Granulocytes

ABSTRACT Classical twin studies and recent linkage analyses of African populations have revealed a potential involvement of host genetic factors in susceptibility or resistance to Mycobacterium tuberculosis infection. In order to identify the candidate genes involved and test their causal implication, we capitalized on the mouse model of tuberculosis, since inbred mouse strains also differ substantially in their susceptibility to infection. Two susceptible and two resistant mouse strains were aerogenically infected with 1,000 CFU of M. tuberculosis, and the regulation of gene expression was examined by Affymetrix GeneChip U74A array with total lung RNA 2 and 4 weeks postinfection. Four weeks after infection, 96 genes, many of which are involved in inflammatory cell recruitment and activation, were regulated in common. One hundred seven genes were differentially regulated in susceptible mouse strains, whereas 43 genes were differentially expressed only in resistant mice. Data mining revealed a bias towards the expression of genes involved in granulocyte pathophysiology in susceptible mice, such as an upregulation of those for the neutrophil chemoattractant LIX (CXCL5), interleukin 17 receptor, phosphoinositide kinase 3 delta, or gamma interferon-inducible protein 10. Following M. tuberculosis challenge in both airways or peritoneum, granulocytes were recruited significantly faster and at higher numbers in susceptible than in resistant mice. When granulocytes were efficiently depleted by either of two regimens at the onset of infection, only susceptible mice survived aerosol challenge with M. tuberculosis significantly longer than control mice. We conclude that initially enhanced recruitment of granulocytes contributes to susceptibility to tuberculosis.

Lipoprotein processing is required for virulence of Mycobacterium tuberculosis

Lipoproteins are a subgroup of secreted bacterial proteins characterized by a lipidated N‐terminus, processing of which is mediated by the consecutive activity of prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (LspA). The study of LspA function has been limited mainly to non‐pathogenic microorganisms. To study a potential role for LspA in the pathogenesis of bacterial infections, we have disrupted lspA by allelic replacement in Mycobacterium tuberculosis, one of the worlds most devastating pathogens. Despite the presence of an impermeable lipid outer layer, it was found that LspA was dispensable for growth under in vitro culture conditions. In contrast, the mutant was markedly attenuated in virulence models of tuberculosis. Our findings establish lipoprotein metabolism as a major virulence determinant of tuberculosis and define a role for lipoprotein processing in bacterial pathogenesis. In addition, these results hint at a promising new target for therapeutic intervention, as a highly specific inhibitor of bacterial lipoprotein signal peptidases is available.

Common and Unique Gene Expression Signatures of Human Macrophages in Response to Four Strains of Mycobacterium avium That Differ in Their Growth and Persistence Characteristics

ABSTRACT Classification of pathogenic species according to the distinct host transcriptional responses that they elicit may become a relevant tool for microarray-based diagnosis of infection. Individual strains of Mycobacterium avium, an opportunistic pathogen in humans, have previously been shown to differ in terms of growth and persistence. In order to cover a wide spectrum of virulence, we selected four M. avium isolates (2151SmO, 2151SmT, SE01, TMC724) that have distinct intramacrophage replication characteristics and cause differential activation in human macrophages. Following infection with each of these strains, the expression of 12,558 genes in human macrophages was systematically analyzed by microarray technology. Fifty genes (including genes encoding proinflammatory cytokines, chemokines, signaling, and adhesion molecules) were differentially expressed more than twofold in response to all of the M. avium isolates investigated and therefore constitute a common macrophage signature in response to M. avium. The magnitude of regulation of most of these genes was directly correlated with the host cell-activating capacity of the particular M. avium strain. The regulation of a number of genes not previously associated with mycobacterial infections was apparent; these genes included genes encoding lymphocyte antigen 64 and myosin X. In addition, individual response patterns typical for some M. avium isolates could be defined by the pronounced upregulation of interleukin-12p40 (IL-12p40) (in the case of 2151SmO) or the specific upregulation of SOCS-1 and IL-10 (in the case of SE01) in macrophages. TMC724, a strain of avian origin, could not be classified by any one of these schemes, possibly indicating the limits of pathogen categorization solely by immune response signatures.

Expression of the ompATb operon accelerates ammonia secretion and adaptation of Mycobacterium tuberculosis to acidic environments

Homeostasis of intracellular pH is a trait critical for survival of Mycobacterium tuberculosis in macrophages. However, mechanisms by which M. tuberculosis adapts to acidic environments are poorly understood. In this study, we analysed the physiological functions of OmpATb, a surface‐accessible protein of M. tuberculosis. OmpATb did not complement the permeability defects of a Mycobacterium smegmatis porin mutant to glucose, serine and glycerol, in contrast to the porin MspA. Uptake rates of these solutes were unchanged in an ompATb operon mutant of M. tuberculosis indicating that OmpATb is not a general porin. Chemical analysis of low‐pH culture filtrates showed that the proteins encoded by the ompATb operon are involved in generating a rapid ammonia burst, which neutralized medium pH and preceded exponential growth of M. tuberculosis. Addition of ammonia accelerated growth of the ompATb operon mutant demonstrating that ammonia secretion is indeed a mechanism by which M. tuberculosis neutralizes acidic environments. Infection experiments revealed that the ompATb operon was not required for full virulence in mice suggesting that M. tuberculosis has multiple mechanisms of resisting phagosomal acidification. Taken together, these results show that the ompATb operon is necessary for rapid ammonia secretion and adaptation of M. tuberculosis to acidic environments in vitro but not in mice.

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest

The success of Mycobacterium tuberculosis depends on its ability to survive within host macrophages. Here, M. tuberculosis avoids the acidic, hydrolytically competent environment of the phagolysosome by arresting phagosome maturation. Having shown previously that a M. tuberculosis mutant deficient in lipoprotein signal peptidase (LspA) is strongly attenuated in vivo in a mouse model of infection, we now studied putative mechanisms involved in attenuation of the lspA : : aph mutant at a cellular level. In this work we investigated the ability of the mutant to interfere with two host defence mechanisms, i.e. Toll-like receptor (TLR)2-dependent immune response and phagosome maturation. While mycobacterial lipoproteins have been reported to trigger a TLR2 signalling pathway critical for innate immune responses, we found that growth control of the lspA : : aph mutant was independent of TLR2. In addition, the lspA : : aph mutant arrested phagosome maturation to an extent similar to that of the wild-type, as measured by lysosomal-associated membrane protein 1 (LAMP1) co-localization and intraphagosomal pH. These observations demonstrate severe attenuation even in the presence of arrested phagosome maturation, and point to a role for the early phagosome in growth restriction of the M. tuberculosis lspA mutant.

Interaction of Rv1625c, a mycobacterial class IIIa adenylyl cyclase, with a mammalian congener

The adenylyl cyclase Rv1625c from Mycobacterium tuberculosis codes for a protein with six transmembrane spans and a catalytic domain, i.e. it corresponds to one half of the pseudoheterodimeric mammalian adenylyl cyclases (ACs). Rv1625c is active as a homodimer. We investigated the role of the Rv1625c membrane domain and demonstrate that it efficiently dimerizes the protein resulting in a 7.5‐fold drop in Km for ATP. Next, we generated a duplicated Rv1625c AC dimer by a head‐to‐tail concatenation. This produced an AC with a domain order exactly as the mammalian pseudoheterodimers. It displayed positive cooperativity and a 60% increase of vmax compared with the Rv1625c monomer. Further, we probed the compatibility of mycobacterial and mammalian membrane domains. The second membrane anchor in the Rv1625c concatamer was replaced with membrane domain I or II of rabbit type V AC. The mycobacterial and either mammalian membrane domains are compatible with each other and both recombinant proteins are active. A M. tuberculosis Rv1625c knockout strain was assayed in a mouse infection model. In vitro growth characteristics and in vivo organ infection and mortality were unaltered in the knockout strain indicating that AC Rv1625c alone is not a virulence factor.

The impact of body mass index and gender on the development of infectious complications in polytrauma patients

PurposeThe aim was to test the impact of body mass index (BMI) and gender on infectious complications after polytrauma.MethodsA total of 651 patients were included in this retrospective study, with an Injury Severity Score (ISS)xa0≥16 and agexa0≥16xa0years. The sample was subdivided into three groups: BMIxa0<25xa0kg/m2, BMI 25–30xa0kg/m2, and BMIxa0>30xa0kg/m2, and a female and a male group. Infectious complications were observed for 31xa0days after admission. Data are given as meanxa0±xa0standard errors of the means. Analysis of variance, Kruskal–Wallis test, χ2 tests, and Pearson’s correlation were used for the analyses and the significance level was set at Pxa0<xa00.05.ResultsThe overall infection rates were 31.0xa0% in the BMIxa0<25xa0kg/m2 group, 29.0xa0% in the BMI 25–30xa0kg/m2 group, and 24.5xa0% in the BMIxa0>30xa0kg/m2 group (Pxa0=xa00.519). The female patients developed significantly fewer infectious complications than the male patients (26.8 vs. 73.2xa0%; Pxa0<xa00.001). The incidence of death was significantly decreased according to the BMI group (8.8 vs. 7.2 vs. 1.5xa0%; Pxa0<xa00.0001) and the female population had a significantly lower mortality rate (4.1 vs. 13.4xa0%; Pxa0<xa00.0001). Pearson’s correlations between the Abbreviated Injury Scale (AIS) score and the corresponding infectious foci were not significant.ConclusionHigher BMI seems to be protective against polytrauma-associated death but not polytrauma-associated infections, and female gender protects against both polytrauma-associated infections and death. Understanding gender-specific immunomodulation could improve the outcome of polytrauma patients.

The myometrial contractility during late pregnancy in dairy cows, in vitro

This study aimed to investigate the in vitro contractility of the myometrium and its relationship to the blood concentrations of estradiol-17β (E2β), progesterone (P4), 15-keto-13,14-dihydro-PGF2α (PGFM) and ionised calcium (Ca2+) prior to tissue harvest in 12 healthy Holstein-Friesian cows in late pregnancy. Three circular (CM) and 3 longitudinal myometrial (LM) strips were dissected during a caesarean section and mounted in an organ bath containing modified Krebs solution (KS). The spontaneous contractility was recorded during five 30-min time periods (T1 to T5), after which the strips were exposed to increasing concentrations of oxytocin (OT; 10-10-10-7M), a natural PGF2α-analogue (PGF; 10-7-10-4M) and KS (Cont) for four 30-min time periods (T6 to T9). The variables area under the curve (AUC), mean (MA) and maximal amplitude (maxA) were calculated for each T. The blood P4, E2ß, Ca2+ and PGFM values averaged 4.0±1.7ng/mL, 482.3±63.7 pg/mL, 0.8±0.3 mmol/L and 125.3±63.7pg/mL. The LM strips had greater AUC, MA, and maxA than CM, and OT caused greater AUC and MA in both muscle layers than PGF or control treatment (OT>PGF>Cont). Estradiol-17β correlated with AUC and MA of LM at T1 to T5 (r=0.69; P≤0.05). In conclusion, LM and CM strips have different contractile performance but show enhanced activity when stimulated with OT and less activity after PGF stimulation if compared with Cont. Blood concentrations of E2β may be useful as an indicator of uterine contractile performance in late pregnant cattle.