aureus virulence in silkworms. The LD50 values of the hla-disrupted mutant, hlb-disrupted mutant, hla/hlb double-disrupted mutant, psmα-deleted mutant and psmβ-deleted mutant were similar to those of the BMN 673 parent strain (Table 4). Thus, hla, hlb, psmα and psmβ encoding hemolysins do not contribute to S. aureus virulence in silkworms. In contrast, the LD50 of the agr mutant was 2.5-fold higher than that of the parent strain (Table 4). This confirms previous findings that the agr locus

contributes to S. aureus virulence in silkworms, and suggests that the agr locus functions in silkworms via hla-, hlb-, psmα- and psmβ-independent pathways. Staphylococcus aureus possesses 16 two-component regulatory systems (Cheung et al., 2004). Among them, arlRS and saeRS broadly regulate the expression of virulence genes (Fournier et al., 2001; Liang et al., 2005, 2006). The arlRS-deleted mutant exhibited attenuated virulence in a mouse systemic infection model (Benton et al., 2004). The saeRS-deleted mutant showed attenuated virulence in a mouse pyelonephritis infection model (Liang et al., 2006). We examined whether the arlS buy LGK-974 and saeS genes of S. aureus contribute to virulence against silkworms. The LD50 values of the arlS- and saeS-disrupted mutants were 2.7- and 1.8-fold higher than

that of the parent strain, respectively (Table 4). This indicates that arlS and saeS contribute to virulence of S. aureus against silkworms. Cell-wall-anchored proteins of S. aureus are reported to contribute to virulence by facilitating bacterial attachment to host tissues or escape from immune systems (Foster Phosphoprotein phosphatase & Hook, 1998). Sortase A is required for anchoring of various proteins to the cell wall (Mazmanian et al., 1999). A gene-disrupted mutant of srtA encoding sortase A had attenuated virulence in mouse infection models (Table 3) (Jonsson et al., 2002, 2003; Weiss et al., 2004). We tested whether the srtA-disrupted mutant showed decreased virulence in silkworms.

The LD50 of the srtA-disrupted mutant was 3.1-fold higher than that of the parent strain (Table 4). This suggests that the anchoring of cell-wall proteins by sortase A is required for S. aureus virulence in silkworms. Mouse pneumonia (Bubeck Wardenburg et al., 2007) Rabbit corneal infection (O’Callaghan et al., 1997) psmα1 psmα2 psmα3 psmα4 PSMα1, PSMα2, PSMα3, PSMα4 Mouse systemic infection (Wang et al., 2007) Mouse skin infection (Wang et al., 2007) psmβ1 psmβ2 PSMβ1, SMβ2 AgrA, AgrB, AgrC, AgrD, RNAIII SA1842 SA1843 SA1844 Mouse pneumonia (Heyer et al., 2002) Rabbit corneal infection (O’Callaghan et al., 1997) Silkworm (Kaito et al., 2005) C. elegans (Sifri et al., 2003) Manduca sexta (Fleming et al., 2006) NA in Drosophila (Needham et al., 2004) SA1246 SA1247 SA1248 SA0660 SA0661 C. elegans (Bae et al.

For instance, in many HIV-infected cohorts, cigarette smoking, recreational drug use (including cocaine use), increased alcohol intake and reduced physical activity are highly prevalent [11]. These factors may also affect the risk of neurocognitive disorders (HIV-associated neurocognitive disease and dementia), non-AIDS-associated

malignancies, liver disease, diabetes, and renal and osteoporotic bone diseases. Some SCH772984 order cohort studies have already suggested that modification of risk factors can decrease the incidence of non-AIDS-defining chronic conditions, including CVD [6]. Hence, it is important to screen and manage risk factors for long-term age-related diseases that increasingly affect the HIV-infected population. Most studies that have examined the contribution of HIV infection to mortality, including those discussed above, do not have an ideal control population. Hence, considerable caution needs to be exercised when attributing relative risk of mortality caused by HIV itself as opposed to unattributed associated confounding variables, particularly lifestyle factors. Even a supposedly ideal control population, such as individuals at high risk of HIV infection but who remain uninfected, might differ in terms of host

factors that govern both infectability and mortality. A study from Denmark that carefully matched cases and controls concluded that mortality in patients without risk factors on successful Selleck Avasimibe HAART therapy is almost identical to that of the non-HIV-infected population [12]. It is important to further define the relationship between HIV infection and mortality, especially those factors that can be modified to attenuate any risk. Screening tools and risk calculators for the general population have been developed for some common noncommunicable chronic diseases, as best exemplified

by coronary heart disease (CHD), fragility fractures, diabetes and renal disease. Personalized risk prediction aims to estimate, communicate and monitor risk to motivate adherence to lifestyle change or therapies, and to allocate scarce prevention Tobramycin resources and strategies appropriately. The World Health Organization (WHO) has recently focused on noncommunicable diseases (NCDs), as they are the leading cause of death globally, killing more people each year than all other causes combined [13]. The WHO has recognized that, contrary to popular opinion, available data demonstrate that nearly 80% of NCD deaths occur in low- and middle-income countries [13]. CVD is one of the leading causes of death in the UK and is largely preventable [14]. In 2008, there were more than 191 000 deaths attributable to heart and circulatory disease in the UK, including 88 000 deaths from CHD and a further 43 000 from stroke.

2). For the phylogenetic analysis, different T4-type phages and g23 clones of marine and terrestrial environments from referred marine and paddy T4 subgroups (Filée et al., 2005; Wang et al., 2009a, b) Z-VAD-FMK clinical trial including the closest relative clones were used. The Bayesian tree obtained in our study is shown in Fig. 3. Our results revealed that neither of the Lake Baikal sequences was grouped into T-evens, PseudoT-evens or SchizoT-evens. The majority of g23 clones from Lake Baikal formed nine deep-branching clusters (B1–B9) with reliable support (79–100%). Two Lake Baikal clusters (B3 and B4) belonged to the ExoT-evens group of marine cyanophages. Clusters B1, B5 and four separate Lake Baikal clones

were grouped with marine or paddy soil T4 subgroups

(Marine groups III, IV; Paddy groups III, VI, VII). The rest of the Baikalian clusters (B2, B6–B9) were separate and the accessory of these clusters to any referred T4-type phage subgroups has not been determined. The most unique sequence found in our study was a clone S0508/1-1. It was clustered with two clones from Japanese paddy fields (KuCf-Jun12-17 and Ch-Cf-Sep22-11) obtained by Wang et al. (2009a). Apparently, this Ixazomib datasheet sequence had originated from an ancestor other than Lake Baikal phage sequences (Fig. 3). In this study, we analyzed the diversity of the T4-type bacteriophages in Northern and Southern Baikal using a PCR strategy based on the partial sequencing Reverse transcriptase of the g23 gene. We also compared these data with the composition and abundance of autotrophic picocyanobacteria and heterotrophic bacteria that are the most probable hosts for T4-like phages. We found that the populations of both bacterial and autotrophic plankton in Northern and Southern Baikal basins were significantly different. Northern Baikal was characterized by a high level of picocyanobacterial development. In contrast to this basin, the predominant numbers of heterotrophic bacteria were registered in Southern Baikal. The differences

in phytoplankton biomass were also recorded, and so the abundance of phytoplankton in Southern Baikal was much higher (Sakirko et al., 2009). Our study showed differences between the sequences of the T4 g23 gene obtained from Northern and Southern Baikal. Five Lake Baikal clusters (B1–B4, B7) were mainly composed of clones from the Northern basin while B5, B6 and B8 generally included clones from the Southern basin. Recently, Sandaa & Larsen (2006) demonstrated pronounced seasonal dynamics of the viral populations in Norwegian coastal waters and showed its correlation with the changes in the abundance of possible hosts. Following from this, we supposed that the biodiversity and quantity of bacterial plankton, autotrophic plankton and phytoplankton in two basins of Lake Baikal have determined a structure of viral communities in general and T4 bacteriophages in particular.

The stained slides were analyzed with a Leica Microscope at 1000× magnification. Pure bacterial clones were stored at −80 °C. Bacterial genome DNA was isolated by applying DNA Mini and Blood Mini

Kit from Qiagen (Hilden, Germany). Freshly subcultured single colonies were harvested with sterile wooden stick cotton swaps and resuspended in PBS. After centrifugation, the pellet was lysed in lysis buffer containing proteinase K provided by the manufacturer. In case of Gram-positive Selleckchem Navitoclax bacteria, lysozyme (20 mg mL−1) was added as recommended by the manufacturer. In brief, the bacterial DNA was isolated by adhering to silicate in mini columns and eluted with water after washing with an ethanol-containing solution. The DNA concentration was measured AZD2281 datasheet with a Nanodrop photometric apparatus (Peqlab, Erlangen, Germany). Purified bacterial genomic DNA was used to amplify a fragment of 1500 bp of the 16S rRNA gene by polymerase chain reaction (PCR) with the forward primer 8F 5′-AGAGTTTGATCCTGGCTCAG-3′ (Galkiewicz & Kellogg, 2008)

and reverse primer DG74 5′-AGGAGGTGATCCAACCGCA-3′ (Greisen et al., 1994) (Eurofins, Ebersberg, Germany). The PCR (25 μL) contained 1 U Dream Taq DNA Polymerase (Fermentas, St. Leon-Roth, Germany), 1× Dream Taq Buffer, 0.5 mM dNTPs, 0.15 μM forward and reverse primer, and 30–50 ng genomic DNA. The PCR mixture was subjected to an initial denaturation step of 5 min at 95 °C, followed by 35 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 52 °C,

and extension of 2 min at 72 °C, and a final extension of 10 min at 72 °C in a Peltier Thermal Cycler PTC-200 (BioRad, Vienna, Austria). The amplification product was visualized by agarose gel electrophoresis (1% agarose in 1× TAE-buffer (40 mM Tris, 20 mM sodium acetate, 1 mM EDTA, pH 8.0)). Midori green (Fermentas) Adenosine triphosphate stained DNA bands (1.5 kb) were excised under a 360-nm UV light box and purified with the NucleoSpin Extract II Kit (Macherey-Nagel, Dueren, Germany). The sequencing of both strands of the amplified 16S rRNA gene was run by Eurofins sending 150 ng of the purified PCR product. The quality of the obtained sequence was checked by screening the chromatogram of each read. The complete sequence was then compared to the DNA databases using the program blast (http://www.ncbi.nlm.nih.gov). Sequence alignments with the highest score were investigated for identifying the bacterial strain by specific 16S rRNA gene sequence. The total bacterial count of each pork meat juice sample is summarized in Table 1 ranging from 104 to 108 CFU mL−1 after 6 h storage at 4 °C. Only 30% of the analyzed samples reached a bacterial load between 107 and 108 CFU mL−1. The results did not reveal any differences between the bacterial count of juice from VP pork meat and in air stored ones.

To determine the relationship between PhoB and the general stress response in V. cholerae, we compared the ability of isogenic wild type, ΔphoB and ΔrpoS mutants to withstand environmental stresses. In Fig. 5 we show that, while strain SZS007 (wild type) and its isogenic ΔrpoS were similarly sensitive to 0.5 mM hydrogen peroxide, the ΔphoB was significantly more sensitive to this level of oxidative stress. The ΔrpoS mutant was more sensitive

than the wild type to higher hydrogen peroxide concentrations (data not shown). This result indicates that expression of PhoB provides protection to oxidative stress by a mechanism different from RpoS. As expected from previous studies (Yildiz & Schoolnik, buy Anti-diabetic Compound Library 1998; Jahid et al., 2006), the ΔrpoS mutant was more sensitive than the wild type to 1.2 M NaCl, while the ΔphoB mutant was more resistant (Fig. 5). The ΔphoB mutant was also more resistant than the wild-type strain and the ΔrpoS mutant to pH 4.5. Finally, the ΔrpoS mutant was more sensitive DNA Damage inhibitor to carbon starvation

compared with wild type and ΔphoB that behaved similarly with regard to this stress. Taken together, the above data are consistent with PhoB modulating environmental stress in a manner independent of the general stress response regulator RpoS. The finding that V. cholerae builds large poly-P stores and our previous observation that a mutant impaired in poly-P biosynthesis was more sensitive to environmental stressors in low-phosphate medium (Ogawa et al., 2000; Jahid et al., 2006) points toward phosphate starvation as a critical environmental stress that could impact the survival of V. cholerae in its aquatic Monoiodotyrosine habitat. Unfortunately, very little is known about how phosphate starvation affects V. cholerae behavior and life style. Extracellular orthophosphate is the major source of

high-energy phosphate for biosynthesis and many signal transduction pathways such as quorum sensing. Therefore, it was not surprising that phosphate limitation enhanced HapR expression, which is repressed when high-energy phosphate is transferred to LuxO. Phosphate starvation is known to activate PhoB and induce the transcription of the PhoB regulon (Lamarche et al., 2008). The fact that HapR represses biofilm formation (Waters et al., 2008) and PhoB has been shown to negatively affect biofilm formation in other Gram-negative bacteria (Monds et al., 2001, 2007) prompted us to examine the relationship between HapR, PhoB and biofilm formation. To this end, we constructed a ΔphoB mutant of strain SZS007. We did not observe any phenotype for this mutant in LB or high-phosphate medium (i.e. growth rate, motility, extracellular protease production). However, the mutant did exhibit reduced growth and alkaline phosphatase expression in low-phosphate medium containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate (data not shown).

7,8,27 Furthermore, bronchoconstriction at low barometric RAD001 mouse pressure exacerbates hypoxia and thus theoretically predisposes asthmatics to HAPE and AMS.2 At altitudes up to 2,000 m, asthmatic travelers receive the benefits of decreased airborne allergens and reduced resistance to airflow.7,8,27,61 At altitudes

above 2,500 m, conditions may be more conducive to induce an asthma attack due to the cold, dry air.61 Travelers at highest risk are those who use inhaled bronchodilators more than three times per week at their living altitude and those who participate in strenuous aerobic activity at altitude.61,62 Between 3,500 and 5,000 m, it has been shown that asthmatics have a reduced risk of suffering an asthma attack. Whereas the cold, dry air provides a stimulus for an asthma attack, changes in physiologic mediators that occur with acclimatization are thought to exert a modulatory effect over airway hyperresponsiveness.7,61,63 While at altitude, use of volumetric spacers is recommended for metered dose BTK inhibitors high throughput screening inhalers, and the mouth should be protected against cold and wind.8,61 It is notable that high altitude natives routinely use silk scarves to protect their airways from exposure to cold air. Exertion at altitude should be moderate to avoid excessive hyperventilation and passive ascent to high altitude should be avoided as sudden exposure to hypoxia can increase airway irritability.61,64 Peak expiratory flow rate is a practical

method for monitoring asthmatic status at PI-1840 altitude.8 Hypobaric hypoxia associated with high altitude is likely to exacerbate the effects of obstructive sleep apnea (OSA). Richalet and colleagues suggest that individuals with Down syndrome and OSA have significantly impaired chemoreceptor sensitivity to hypoxia and are thus at increased risk of HAPE with exposure to even moderate altitudes.65 Thus, high altitude travel is contraindicated for people with OSA who demonstrate arterial oxygen desaturation at sea level.31 It is of interest that

acetazolamide has been shown to reduce the apnea–hypopnea index in patients with OSA.66 Should a patient with OSA choose to travel to altitude, it is reasonable to prescribe acetazolamide prophylaxis in an effort to improve the symptoms of OSA and reduce the risk of developing AMS. Patients who travel with their continuous positive airway pressure machine may need to adjust the pressure setting to accommodate for the decrease in barometric pressure at altitude.8 No baseline data exist to help the physician predict which patients with interstitial lung disease (ILD) are most likely to suffer deterioration in their respiratory status at high altitude. It is recommended that patients with ILD in whom the presence of pulmonary hypertension has not been confirmed should undergo echocardiography before traveling to high altitude. Symptomatic pulmonary hypertension is a contraindication to high altitude travel.

Infrequent diagnoses (those

with a frequency of <10 cases) were also recorded. Continuous variables were expressed as the mean and standard deviation when normally distributed, as the median and interquartile range (IQR) if distribution was skewed, and discrete variables as percentages. The Student's independent samples t-test was used to compare continuous variables and the Mann–Whitney U-test for continuous variables without a normal distribution. The association between categorical variables was evaluated using a chi-squared test (when samples were of sufficient size) or with a Fisher's exact test. Magnitude of the buy PFT�� effect was expressed as a 95% confidence interval. A p value of <0.005 was considered statistically

significant. A total of 2,993 travelers were included in the study; 11 of them were excluded because destination did not correspond with the areas included in the study. The total number of travelers analyzed was 2,982. In total, 47.8% were women; median age was 35 years (IQR 28 to 40). Median time elapsed from return to consultation was 30 days (IQR 13 to 90). Geographical areas of travel and number of travelers to each area are shown in Figure 1. The duration of travel in order of frequency was: short term in 1,594 (53.4%), long term in 710 (23.8%), and medium term in 678 (22.7%) cases. The type of travel in order of frequency was: type A in 979 (32.8%), type B in 511 (17.1%), type C in 508 (17%), and type D in 984 (33%) mTOR inhibitor cases. The age of the traveler, duration, and type of travel depending on the geographic area visited are shown in Table 1. In total, 2,062 had received a travel-related vaccine (69.1%), and the median number of vaccines received was two (IQR: 1 to

4). In order of frequency, vaccines received were: yellow fever (79.1%), typhoid fever (55.9%), tetanus–diphtheria (44%), hepatitis B (40.6%), and hepatitis A (31.8%). Complete information was available regarding malarial chemoprophylaxis in 2,568 (86.08%) cases. In total, 1,059 (35.5%) had taken malarial chemoprophylaxis, with variations according to geographical area of travel: prophylaxis was used by 54.4% of travelers to sub-Saharan Africa, 33.5% to Central Asia Southeast, 19.4% to South America, 11.5% these to the Caribbean–Central America, and 5.1% to other destinations (p < 0.05). Of these 1,059, 623 (58.8%) took chemoprophylaxis correctly. This proportion varied depending on the drug used: 57 of 71 (80.3%) taking atovaquone–proguanil did so correctly, 274 of 409 (67%) taking mefloquine, 23 of 43 (53.5%) taking doxycycline, 193 of 379 (50.9%) taking chloroquine–proguanil, and 85 of 176 (48.3%) taking chloroquine; χ2 = 43.3 (p < 0.001). More than 75% of the cases had one of the following five presenting syndromes: 1,028 (34.5%) febrile syndrome, 872 (29.

Western blot analysis of SLP-2 transfected cells as well as control transfected cells was performed as described above. Mouse brain homogenates were purified using a differential centrifugation method described earlier (Rosenthal et al., 1987; Moreira et al., 2003). For isolation of mitochondria-enriched fractions, adult female CD-1 mice (n = 11) were killed, and the cerebral cortex was immediately dissected and put on ice. Tissue was manually homogenized at

+4 °C in 10 mL isolation buffer [in mm: mannitol, 225; sucrose, 75; HEPES, 5; EGTA, 1; bovine serum albumin (BSA), 1 mg/mL; protease type VIII, 0.3125 mg/mL from Sigma–Aldrich at pH 7.4] and centrifuged at 2000 g for 5 min. The pellet, including the synaptosomal

layer, was resuspended in isolation buffer now containing 0.02% (w/v) digitonin and centrifuged at 12 000 g for 10 min. The pellet without the synaptosomal Anti-diabetic Compound Library datasheet layer was resuspended in isolation buffer and centrifuged Doxorubicin datasheet at 12 000 g for 10 min. The pellet was finally resuspended in 100 μL of resuspension buffer (in mm: mannitol, 225; sucrose, 75; HEPES, 5; at pH 7.4). For isolation of synaptosome-enriched fractions, adult female CD-1 mice (n = 4) were used, and the cerebral cortex was homogenized in 1.5 mL of isolation buffer [in mm: mannitol, 215; sucrose, 75; HEPES, 20; EGTA, 1; 0.1% (w/v) fatty acid-free BSA at pH 7.2] followed by centrifugation at 1300 g for 3 min. The supernatant was removed and the resuspended pellets were again

centrifuged at 1300 g for 3 min. The two sets of supernatants were pooled, topped off with isolation buffer and centrifuged at Monoiodotyrosine 13 000 g for 10 min. The supernatant was discarded; the pellet was resuspended in isolation buffer and centrifuged at 10 000 g for 10 min. The pellet was resuspended in isolation buffer without EGTA and centrifuged at 10 000 g for 10 min. The final cell fragments containing pellet were resuspended in 50 μL of isolation buffer without EGTA. For mitochondrial respiration analyses, 0.5 mg/mL protein was added into the reaction chamber of a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK) set to +37 °C and filled with 1 mL respiration buffer (in mm: sucrose, 100; HEPES, 5; KCl, 100; KH2PO4, 2; EGTA, 10 μm; pH 7.4). Prior to application of oxidative substrates, WIN 55,212-2 (WIN; Biomol International LP, Plymouth, PA, USA; final concentration 0.05 μm), which was pre-dissolved in dimethyl sulfoxide (DMSO; stock solution 100 mm; stored at −20 °C), or DMSO alone diluted in respiration buffer (1 : 2000) were added into the reaction chamber. Thirty seconds later, pyruvate (5 mm) and malate (2.5 mm) were added concomitantly as the oxidative substrates. To determine ADP-dependent respiration (complex III activity), ADP (2.5 mm) was added.

It PLX4032 order was also observed that probiotic

dahi suppressed the diabetes progression and its complication through enhancing antioxidant system (Yadav et al., 2008). Though, the actual link between probiotic-mediated pathology of obesity and diabetes has been debated on the basis of farm animal’s data (Raoult, 2008; Delzenne & Reid, 2009; Ehrlich, 2009). In relation to these controversies, Bifidobacteria, one of the important classes of probiotic organisms, have been found to be decreased in overweight women in comparison with normal weight women (Santacruz et al., 2009). Recent studies have suggested that probiotic-based selective strains of Lactobacilli and Bifidobacteria show beneficial effects on obesity and type-2 diabetes (Aronsson et al., 2010). Andreasen et al. (2010) reported that L. acidophilus decreased the insulin resistance and inflammatory markers in human see more subjects. More recently, Vajro et al. (2011) and others (Kang et al., 2010; An et al., 2011; Chen et al., 2011; Naito

et al., 2011) showed that feeding of specific strains of Lactobacilli and Bifidobacteria ameliorate the progression of obesity and diabetes, suggesting that probiotic-mediated modulation of gut flora can be a potential therapy against obesity and diabetes. Although animal studies have shown promising results in probiotic-mediated tetracosactide suppression of obesity and diabetes, very few studies in humans showed the significant effects. Hence, it is required to conduct well-designed studies for examining the efficacy of probiotic-based

formulation in the treatment for obesity and diabetes. Also, the mechanism(s) of action for probiotic-based formulation is not completely understood; therefore, future studies should also be focused on describing the probiotic action–targeted molecules and organs in physiologic models. Certain functional foods containing probiotic provide preformed lactase to gut and allow better digestion of lactose. The regulatory role of probiotics in allergic disease was demonstrated by a suppressive effect on lymphocytes’ proliferation and interleukin-4 generation in vitro (Sutas et al., 1996). Subsequently, the immune inflammatory responses to dietary antigens in allergic individuals were shown to be alleviated by probiotics, this being partly attributable to enhance the production of anti-inflammatory cytokines (Pessi et al., 2000) and transferring growth factor-β (Haller et al., 2000). Probiotic bacteria also possess prophylactic and therapeutic properties.

2b and c). The hot spring isolates, on the other hand, showed almost no variation; only

3 of the 12 occurring polymorphisms were present, with a preference for 14 repeat units (Fig. 2d). It was also observed that strains that had the same seven-gene SBT could still have a different copy number for the lcl VNTR region (data not shown). As addition of the seventh gene neuA to the previously used six-gene SBT scheme enhanced the discriminatory power (Ratzow et al., 2007), it is possible that additional genes can even further discriminate the presently established SBT types. Therefore, our sequencing data reinforce the observation made by Pourcel et al. (2007) that the VNTR region of lcl (lpms31) is very diverse and could be an additional tool to distinguish among isolates. However, we have to be cautious about its genetic variability. If this genetic variability is extremely large, the chance of PLX3397 in vivo two isolates of the same population being different is almost equal to the selleck screening library chance of two isolates of different locations being different. This problematic genetic variability was previously shown for three other L. pneumophila loci: fliC, proA and mompS (Coscolláet al., 2006). This emphasizes that attention

has to be paid when selecting a gene for discrimination purposes. More specifically, the genetic variability of the gene has to be high enough to discriminate between different isolates, but small enough to be stable in the period between the outbreak and epidemiological typing. Based on the knowledge that very mammalian collagen plays a role in cell adhesion (Kadler

et al., 2007), the role of Lcl in the adhesion of L. pneumophila Philadelphia to host cells was examined. For this, we attempted to prepare an Lcl-negative mutant. Notwithstanding many attempts, using (1) homologous recombination with a resistance cassette with flanking regions of the lcl gene of different sizes; (2) point mutagenesis; and (3) different resistance cassettes and shuttle vectors, a correct insertion was never obtained, even after screening thousands of colonies. Whether the failure of obtaining a deletion mutant is due to the presence of the VNTR regions present in the lcl gene is not known, but VNTR regions are known to have greater instability. Because we were not able to obtain an Lcl-negative mutant, the role of Lcl in L. pneumophila adhesion and invasion of host cells was determined indirectly by comparing the Philadelphia WT cells and the same cells preincubated with Lcl-specific antibodies. The preincubation of 5 × 107L. pneumophila Philadelphia-1 cells with 20 μg Lcl-specific antibodies for 30 min decreased the adhesion to A549 and macrophage-like cells by 59% (P=0.0015) and 39% (P=0.006), respectively (Fig. 3a and b). In contrast, adhesion and invasion of the amoebae A.