Synergistic activity of synthetic N-terminal peptide of human lactoferrin in combination with various antibiotics against carbapenem-resistant Klebsiella pneumoniae strains.

The spread of multi-drug resistant (MDR) Klebsiella pneumoniae strains producing carbapenemases points to a pressing need for new antibacterial agents. To this end, the in-vitro antibacterial activity of a synthetic N-terminal peptide of human lactoferrin, further referred to as hLF1-11, was evaluated against K. pneumoniae strains harboring different carbapenemase genes (i.e. OXA-48, KPC-2, KPC-3, VIM-1), with different susceptibility to colistin and other antibiotics, alone or in combination with conventional antibiotics (gentamicin, tigecycline, rifampicin, clindamycin, and clarithromycin). An antimicrobial peptide susceptibility assay was used to assess the bactericidal activity of hLF1-11 against the different K. pneumoniae strains tested. The synergistic activity was evaluated by a checkerboard titration method, and the fractional inhibitory concentration (FIC) index was calculated for the various combinations. hLF1-11 was more efficient in killing a K. pneumoniae strain susceptible to most antimicrobials (including colistin) than a colistin-susceptible strain and a colistin-resistant MDR K. pneumoniae strain. In addition, hLF1-11 exhibited a synergistic effect with the tested antibiotics against MDR K. pneumoniae strains. The results of this study indicate that resistance to hLF1-11 and colistin are not strictly associated, and suggest an hLF1-11-induced sensitizing effect of K. pneumoniae to antibiotics, especially to hydrophobic antibiotics, which are normally not effective on Gram-negative bacteria. Altogether, these data indicate that hLF1-11 in combination with antibiotics is a promising candidate to treat infections caused by MDR-K. pneumoniae strains.

Interaction of Mycobacterium tuberculosis cell wall components with the human natural killer cell receptors NKp44 and Toll-like receptor 2.

We have previously demonstrated that a soluble form of the human NK cell natural cytotoxicity receptor NKp44, binds to the surface of Mycobacterium tuberculosis (MTB). Herein, we investigated the interaction of MTB cell wall components (CWC) with NKp44 or with Toll-like receptor 2 (TLR2) and the role of NKp44 and TLR2 in the direct activation of NK cells upon stimulation with MTB CWC. By using several purified bacterial CWC in an ELISA, we demonstrated that NKp44 was able to bind to the MTB cell wall core mycolyl-arabinogalactan-peptidoglycan (mAGP) as well as to mycolic acids (MA) and arabinogalactan (AG), while soluble TLR2 bound to MTB peptidoglycan (PG), but not to MA or AG. The mAGP complex induced NK cell expression of CD25, CD69, NKp44 and IFN-γ production at levels comparable to M. bovis Bacillus Calmette-Guérin-stimulated (BCG) cells. While AG and MA used alone failed to induce NK cell activation, mycobacterial PG-exhibited NK cell stimulatory capacity. Activation of resting NK cells by mAGP and IFN-γ production were inhibited by anti-TLR2 MAb, but not by anti-NKp44 MAb. Differently, anti-NKp44 MAb partially inhibited CD69 expression on NK cells pre-activated with IL-2 and then stimulated with mAGP or whole BCG. Overall, these results provide evidence that components abundant in mycobacterial cell wall are able to interact with NKp44 (AG, MA) and TLR-2 (PG), respectively. While interaction of TLR2 with mycobacterial cell wall promotes activation of resting NK cells and IFN-γ production, NKp44 interaction with its putative ligands could play a secondary role in maintaining cell activation.

Human CD56bright and CD56dim natural killer cell subsets respond differentially to direct stimulation with Mycobacterium bovis bacillus Calmette-Guérin.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin-12 and professional antigen presenting cells. To assess the contribution of two main human NK-cell subsets (CD56(dim) and CD56(bright)) to the overall in vitro NK-cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool-adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU(+) cells were found among the CD56(bright) subset than the CD56(dim) subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon-gamma (IFN-gamma) revealed that CD56(bright) cells were those mainly involved in IFN-gamma production in response to BCG. In contrast, the CD56(dim) subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis-mediated cytotoxicity, than the CD56(bright) subset. Although 16-20-h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56(dim), but not in the CD56(bright) subset, following 4-h incubation with the NK-sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG-stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56(dim) subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56(bright) and CD56(dim) human NK-cell subsets exert different functional activities in response to a live bacterial pathogen.

Purification, biochemical characterization and immunogenicity of SA5K, a secretion antigen of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) secretory proteins are generally considered important antigens for immune protection against tuberculosis (TB). An 8.3-kDa secretory antigen of MTB and Mycobacterium bovis bacillus Calmette-Guérin (BCG), called SA5K, was recently identified and cloned in our laboratory. In this report, recombinant SA5K containing a histidine hexamer was expressed in Escherichia coli and purified to investigate its biochemical structure and to establish whether it was immunogenic for healthy sensitized and nonsensitized human donors and for patients infected with MTB. The protein nucleotide sequence was shown to be identical in BCG and in MTB. SA5K revealed an abnormal electrophoretic mobility in SDS-PAGE that made it look lighter than it is in Western blotting. While recombinant SA5K was poorly recognized by T lymphocytes from patients with pulmonary TB, it elicited proliferation of CD4+ T lymphocytes in the vast majority of healthy individuals sensitized to mycobacterial antigens by BCG vaccination. At a serum dilution of 1 : 80, antibodies reacting against recombinant SA5K were found in 67% of sera from TB patients and in 73% of sera from healthy subjects. The percentage of positive subjects dropped at higher serum dilutions, but no significant difference in the recognition rate was observed between TB patients and healthy donors and between healthy vaccinated and nonvaccinated subjects. Owing to the high percentage of sera from healthy subjects who recognized SA5K in Western blotting, the antigen seems to exhibit, at least in the present form, a poor specificity for an employment for a serodiagnosis of TB.

Identification, molecular cloning, and evaluation of potential use of isocitrate dehydrogenase II of Mycobacterium bovis BCG in serodiagnosis of tuberculosis.

Diagnosis of tuberculosis is time-consuming and requires infrastructures which are often not available in countries with high incidences of the disease. In the present study, an 82-kDa protein antigen was isolated by affinity chromatography and was identified by peptide mass fingerprinting as isocitrate dehydrogenase II, which is encoded by the icd2 gene of Mycobacterium bovis BCG. The icd2 gene of BCG was cloned by PCR, and the product of recombinant gene expression was purified and analyzed by two-dimensional polyacrylamide gel electrophoresis. The recombinant protein, named rICD2, was tested for its recognition by immunoglobulin G (IgG) antibodies from the sera of 16 patients with tuberculosis (TB) and 23 healthy individuals by Western blotting. The results showed that rICD2 is recognized by IgG antibodies from the sera of all TB patients tested at serum dilutions of > or = 1:640. At a serum dilution of 1:1,280, the sensitivity was 50% and the specificity was 86.9%. These results indicate that rICD2 might represent a candidate for use in a new assay for the serodiagnosis of TB.

Involvement of the Mycobacterium tuberculosis secreted antigen SA-5K in intracellular survival of recombinant Mycobacterium smegmatis.

A new protein (SA-5K) secreted in culture filtrates by Mycobacterium bovis, Mycobacterium tuberculosis, and few other mycobacterial species was previously identified and purified in our laboratory. In order to evaluate the putative role of SA-5K during intracellular mycobacterial growth, in the present study the SA-5K gene was cloned and expressed in Mycobacterium smegmatis, a rapid growing non-pathogenic mycobacterium which does not contain the gene for the protein. SA-5K expression in the THP-1 human macrophage cell line infected with the recombinant strain (M. smegmatis-pROL5K) was demonstrated by RT-PCR on RNA extracted from bacterial cells following 24 and 48 h of infection. Intracellular SA5K expression was associated with a higher cfu increase of M. smegmatis-pROL5K in comparison to the negative control strain (M. smegmatis recombinant for the cloning vector) (P=0.01). No significant change in SA-5K synthesis by M. smegmatis-pROL5K was observed when the recombinant strain was grown in vitro in different stress conditions such as iron deprivation, pH 4.5, presence of nitric oxide or hydrogen peroxide. The results presented in this study suggest a possible role for SA-5K in intracellular survival of recombinant M. smegmatis, though the function of the protein remains unknown.

Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection.

Mycobacterium tuberculosis is the infectious agent giving rise to human tuberculosis. The entire genome of M. tuberculosis, comprising approximately 4000 open reading frames, has been sequenced. The huge amount of information released from this project has facilitated proteome analysis of M. tuberculosis. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied to fractions derived from M. tuberculosis culture filtrate, cell wall, and cytosol, resulting in the resolution of 376, 413, and 395 spots, respectively, in silver-stained gels. By microsequencing and immunodetection, 38 culture filtrate proteins were identified and mapped, of which 12 were identified for the first time. In the same manner, 23 cell wall proteins and 19 cytosol proteins were identified and mapped, with 9 and 10, respectively, being novel proteins. One of the novel proteins was not predicted in the genome project, and for four of the identified proteins alternative start codons were suggested. Fourteen of the culture filtrate proteins were proposed to possess signal sequences. Seven of these proteins were microsequenced and the N-terminal sequences obtained confirmed the prediction. The data presented here are an important complement to the genetic information, and the established 2-D PAGE maps (also available at: www.ssi.dk/publichealth/tbimmun) provide a basis for comparative studies of protein expression.

Analysis of the Mycobacterium bovis hsp60 promoter activity in recombinant Mycobacterium avium.

A clinical isolate of Mycobacterium avium was transformed with a new shuttle plasmid containing the Escherichia coli beta-galactosidase reporter gene under the control of the Mycobacterium bovis bacillus Calmette-Guérin (BCG) hsp60 promoter. beta-Galactosidase activity was assayed spectrophotometrically in bacterial homogenates of the recombinant strain (M. avium::lacZ) and used for quantification of the hsp60 promoter strength in different conditions of extra- and intracellular growth. Very low levels of beta-galactosidase were recorded during the exponential phase of in vitro growth, while they increased progressively during the late exponential and stationary phases. A significant increase in enzyme activity was also induced in exponentially growing cells by shifting the incubation temperature from 37 to 45 degrees C, but not from 37 to 42 degrees C nor from 30 to 42 degrees C. No induction of the promoter was observed by adding hydrogen peroxide to the cultures. Finally, beta-galactosidase levels were quantified during growth of M. avium::lacZ in murine macrophages. Soon after phagocytosis and, to a lesser extent at 1, 5 and 7 days after infection, increased levels of bacterial beta-galactosidase were observed indicating an increment in transcriptional activity of hsp60 promoter both at early phases of infection and during the course of intracellular growth.

Identification and molecular cloning of a novel secretion antigen from Mycobacterium tuberculosis and Mycobacterium bovis BCG.

A novel protein called SA-5K was identified in Mycobacterium bovis BCG (BCG) short-term culture filtrates (CFs) by means of a recently described monoclonal antibody (mAb), L8D8. This protein had an apparent molecular mass (MM) of 5 kDa, as judged by Western blotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions, and did not seem to contain any sugar or lipid substituents. In the present work, SA-5K was purified from BCG CFs by affinity chromatography. A protein that could be detected in Western blot but not by standard protein staining techniques was obtained. When SA-5K was subjected to aminoterminal sequencing, the 10 amino acids (aa) found matched the first 10-aa sequence deduced from an open reading frame (ORF) of M. tuberculosis. The ORF encodes a polypeptide, likely to include a signal for secretion, with an estimated MM of 8.3 kDa after signal peptide cleavage. The secretory nature of SA-5K was confirmed by the fact that it could only be detected in CFs, but not in other BCG subcellular fractions. After size exclusion chromatography, reactivity with mAb L8D8 was found to peak in the 45-50- and 14-16-kDa fractions. The latter MM was close to that estimated from the ORF of M. tuberculosis, implying that the 5-kDa antigen detected initially by Western blot in reducing conditions was a portion of SA-5K released after reduction of a disulphide bridge. The presence of the gene for SA-5K in BCG and its identity were confirmed by PCR (polymerase chain reaction) with specific primers and restriction analysis: the PCR product was slightly shorter in BCG than in M. tuberculosis. The gene coding for SA-5K was cloned by PCR from BCG and M. tuberculosis DNA and was expressed in Escherichia coli.

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin.

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.

Comparative analysis of subcellular distribution of protein antigens in Mycobacterium bovis bacillus Calmette-Guérin.

The distribution of protein antigens in purified subcellular fractions of Mycobacterium bovis bacillus Calmette-Guérin (BCG) was comparatively analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with specific monoclonal antibodies and polyclonal sera. The 19- and 38-kDa lipoproteins were mainly detected in the cell wall and cell membrane enriched fractions, and they were extracted from the former by Triton X-114 and Nonidet P-40. The 65-kDa heat-shock protein (hsp) was present in the cytoplasmic fraction and only trace amounts were found in the crude cell wall preparation. In contrast, the 14-kDa hsp was highly represented in the cell wall fraction, besides being present in cytoplasmic fraction. Both superoxide dismutase (SOD) and antigen 85 complex (Ag 85) were abundantly released in culture medium, and to a lower extent, they were present in the cell wall fraction; SOD was present in comparable amounts also in the cytoplasmic fraction, while Ag 85 was far less represented in the same. Sera from mice immunized with culture filtrate (CF) proteins of BCG recognized several antigens in CFs, which were not detectable in cell wall, cell membrane, and cytoplasmic fractions, indicating that CF proteins include secreted antigens which have not yet been identified.