Macrophage Infectivity Potentiator Research Articles

Pharmacophore based virtual screening for identification of marine bioactive compounds as inhibitors against macrophage infectivity potentiator (Mip) protein of Chlamydia trachomatis

Macrophage infectivity potentiator (Mip) is the virulence factor fromChlamydia trachomatisthat is primarily responsible for causing sexually transmitted diseases (STDs) and blindness.

In Silico Analysis of Conformational Changes Induced by Normal and Mutation of Macrophage Infectivity Potentiator Catalytic Residues and its Interactions with Rapamycin.

The Legionella pneumophila (Lp), human pathogen, causes severe and often fatal Legionnaires' disease, produces a major virulence factor, termed 'macrophage infectivity potentiator protein' (Mip), that is necessary for optimal multiplication of the bacteria within human alveolar macrophages. Mip exhibits peptidyl prolyl cis-trans isomerase (PPIase) activity, which can be inhibited by rapamycin and FK506. It was previously shown that substitutions at the catalytic residues, aspartate-142 position replaced to leucine-142 and tyrosine-185 position replaced to alanine-185 strongly reduces the PPIase activityof Mip proteins. Therefore, we aim to develop an in silico mutagenesis model for both important catalytic residues, validated the stability of the mutated model. Further, we have docked the known inhibitor rapamycin with Lp Mip (native) and mutants (D142L and Y185A) to analyze the conformational and binding mode. Electrostatic contributions and van der Waals interactions are the major driving forces for rapamycin binding and largely responsible for the binding differences between the Lp Mip (native and mutated) proteins.

Discovery of Marine Sponge Compound as Promising Inhibitor for Macrophage Infectivity Potentiator (Mip) Protein against Chlamydia pneumoniae

Discovery of Marine Sponge Compound as Promising Inhibitor for Macrophage Infectivity Potentiator (Mip) Protein against Chlamydia pneumoniae

Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila

Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung–epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30–40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.

Influence of FkpA variants on survival and replication of Cronobacter spp. in human macrophages

Members of the genus Cronobacter are responsible for cases of meningitis and bacteremia with high fatality rates in neonates. Macrophage uptake of invading microbes is an innate process, and it has been proposed that macrophage infectivity potentiator (Mip) like proteins enhance the ability of pathogens to survive within macrophages. Cronobacter harbor the mip-like gene fkpA, but its role in intracellular survival of these bacteria in human macrophages has not yet been studied. Application of gentamicin exclusion assays and human THP-1 macrophage cells revealed significant differences in the capablility of Cronobacter species to survive and replicate within macrophages. Analysis to the amino acid level revealed both length and sequence variations in FkpA proteins among species. In this study, we addressed the possible influence of FkpA variants in intracellular survival of Cronobacter spp. in human macrophages, by knocking out the fkpA genes in two different Cronobacter strains and subsequent complementation with variants of the fkpA genes.Our results provide strong evidence that, in Cronobacter spp., FkpA must be considered a virulence factor, but its influence on macrophage survival and replication varies among strains and/or species due to the presence of amino acid variations.

In silico analysis of conformational changes induced by normal and mutation of macrophage infectivity potentiator catalytic residues and its interactions with Rapamycin.

The Legionella pneumophila (Lp), human pathogen causes severe and often fatal Legionnaires' disease, produces a major virulence factor, termed 'macrophage infectivity potentiator protein' (Mip), that is necessary for optimal multiplication of the bacteria within human alveolar macrophages. Mip exhibits peptidyl prolyl cistrans isomerase (PPIase) activity, which can be inhibited by Rapamycin and FK506. Mutation of Mip protein on catalytic residues at Aspartate-142 position replaced to Leucine-142 and Tyrosine-185 position replaced to Alanine-185 that strongly reduces the PPIase activity. Therefore, we aim to develop an in-silico mutagenesis model for both important catalytic residues, validated the stability of the mutated model. Further, we have docked to the known inhibitor rapamycin with Lp Mip (native) and mutants (D142L and Y185A) to analyze the conformational and binding model. For electrostatic contributions and VanderWaals interactions are the major driving force for rapamycin binding and largely responsible for the binding differences between the Lp Mip (native and mutated) proteins.

Structural Genomics Support for Infectious Disease Drug Design.

Structural Genomics Support for Infectious Disease Drug Design.

Map-based comparative genomic analysis of virulent haemophilus parasuis serovars 4 and 5.

Haemophilus parasuis is a commensal bacterium of the upper respiratory tract of healthy pigs. However, in conjunction with viral infections in immunocompromised animals H. parasuis can transform into a pathogen that is responsible for causing Glasser's disease which is typically characterized by fibrinous polyserositis, polyarthritis, meningitis and sometimes acute pneumonia and septicemia in pigs. Haemophilus parasuis serovar 5 is highly virulent and more frequently isolated from respiratory and systemic infection in pigs. Recently a highly virulent H. parasuis serovar 4 was isolated from the tissues of diseased pigs. To understand the differences in virulence and virulence-associated genes between H. parasuis serovar 5 and highly virulent H. parasuis serovar 4 strains, a genomic library was generated by TruSeq preparation and sequenced on Illumina HiSeq 2000 obtaining 50 bp PE reads. A three-way comparative genomic analysis was conducted between two highly virulent H. parasuis serovar 4 strains and H. parasuis serovar 5. Haemophilus parasuis serovar 5 GenBank isolate SH0165 (GenBank accession number CP001321.1) was used as reference strain for assembly.Results of these analysis revealed the highly virulent H. parasuis serovar 4 lacks genes encoding for, glycosyl transferases, polysaccharide biosynthesis protein capD, spore coat polysaccharide biosynthesis protein C, polysaccharide export protein and sialyltransferase which can modify the lipopolysaccharide forming a short-chain LPS lacking O-specific polysaccharide chains often referred to as lipooligosaccharide (LOS). In addition, it can modify the outer membrane protein (OMP) structure. The lack of sialyltransferase significantly reduced the amount of sialic acid incorporated into LOS, a major and essential component of the cell wall and an important virulence determinant. These molecules may be involved in various stages of pathogenesis through molecular mimicry and by causing host cell cytotoxicity, reduced inflammatory and immunological response to infection with this organism. The mechanism by which sialyation of LPS contributes to virulence is a key to understanding the pathogenesis of this highly virulent H. parasuis serovar 4. This analysis also revealed the presence of virulence associated genes similar to the MerR family transcriptional regulators, macrophage infectivity potentiator protein, hemolysin, opacity associated protein, toxin antitoxin system, and virulence associated protein D and colicins. Haemophilus parasuis serovar 4 variants also possess extensive metal ion uptake and regulation mechanism which controls various virulence and virulence associated genes. A combination of virulence associated factors and/or genes and proteins with overlapping functions may be responsible for the apparent enhanced virulence of this organism.The extensive structural modification of LOS and OMP of variant H. parasuis serovar 4 strains appear to aid in nasal colonization, are associated with the organisms' ability to evade the host immune response and provide serum-resistance. In addition, the combination of capsule modification and phase variation due to LOS substitutions could help variant H. parasuis serovar 4 transform into a highly virulent pathogen. Based on these results, the variant H. parasuis serovar 4 strains harbor a diverse repertoire of virulence associated genes which have not been previously reported.

Recombinant protein truncation strategy for inducing bactericidal antibodies to the macrophage infectivity potentiator protein of Neisseria meningitidis and circumventing potential cross-reactivity with human FK506-binding proteins.

A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human proteins, we immunized mice with recombinant truncated type I rMIP proteins that lacked the globular domain and the signal leader peptide (LP) signal sequence (amino acids 1 to 22) and contained the His purification tag at either the N or C terminus (C-term). The immunogenicity of truncated rMIP proteins was compared to that of full (i.e., full-length) rMIP proteins (containing the globular domain) with either an N- or C-terminal His tag and with or without the LP sequence. By comparing the functional murine antibody responses to these various constructs, we determined that C-term His truncated rMIP (-LP) delivered in liposomes induced high levels of antibodies that bound to the surface of wild-type but not Δmip mutant meningococci and showed bactericidal activity against homologous type I MIP (median titers of 128 to 256) and heterologous type II and III (median titers of 256 to 512) strains, thereby providing at least 82% serogroup B strain coverage. In contrast, in constructs lacking the LP, placement of the His tag at the N terminus appeared to abrogate bactericidal activity. The strategy used in this study would obviate any potential concerns regarding the use of MIP antigens for inclusion in bacterial vaccines.

Peptidylprolyl cis-trans isomerases of Legionella pneumophila: virulence, moonlighting and novel therapeutic targets.

Legionella pneumophila, typically a parasite of free-living protozoa, can also replicate in human alveolar macrophages and lung epithelial cells causing Legionnaires' disease in humans, a severe atypical pneumonia. The pathogen encodes six peptidylprolyl cis-trans isomerases (PPIases), which generally accelerate folding of prolyl peptide bonds, and influence protein folding. PPIases can be divided into three classes, cyclophilins, parvulins and FK506-binding proteins (FKBPs). They contribute to a multitude of cellular functions including bacterial virulence. In the present review, we provide an overview of L. pneumophila PPIases, discussing their known and anticipated functions as well as moonlighting phenomena. By taking the example of the macrophage infectivity potentiator (Mip) of L. pneumophila, we highlight the potential of PPIases as promising drug targets.

FipB, an essential virulence factor of Francisella tularensis subsp. tularensis, has dual roles in disulfide bond formation.

FipB, an essential virulence factor of Francisella tularensis, is a lipoprotein with two conserved domains that have similarity to disulfide bond formation A (DsbA) proteins and the amino-terminal dimerization domain of macrophage infectivity potentiator (Mip) proteins, which are proteins with peptidyl-prolyl cis/trans isomerase activity. This combination of conserved domains is unusual, so we further characterized the enzymatic activity and the importance of the Mip domain and lipid modification in virulence. Unlike typical DsbA proteins, which are oxidases, FipB exhibited both oxidase and isomerase activities. FipA, which also shares similarity with Mip proteins, potentiated the isomerase activity of FipB in an in vitro assay and within the bacteria, as measured by increased copper sensitivity. To determine the importance of the Mip domain and lipid modification of FipB, mutants producing FipB proteins that lacked either the Mip domain or the critical cysteine necessary for lipid modification were constructed. Both strains replicated within host cells and retained virulence in mice, though there was some attenuation. FipB formed surface-exposed dimers that were sensitive to dithiothreitol (DTT), dependent on the Mip domain and on at least one cysteine in the active site of the DsbA-like domain. However, these dimers were not essential for virulence, because the Mip deletion mutant, which failed to form dimers, was still able to replicate intracellularly and retained virulence in mice. Thus, the Mip domains of FipB and FipA impart additional isomerase functionality to FipB, but only the DsbA-like domain and oxidase activity are essential for its critical virulence functions.

Legionella spp. in UK composts—a potential public health issue?

Over the past 5 years, a number of cases of legionellosis in Scotland have been associated with compost use; however, studies investigating sources of infection other than water systems remain limited. This study delivers the first comprehensive survey of composts commonly available in the UK for the presence of Legionella species. Twenty-two store-bought composts, one green-waste compost and one home-made compost were tested for Legionella by culture methods on BCYE-α medium, and the findings were confirmed by macrophage infectivity potentiator (mip) speciation. Twenty-two of the samples were retested after an enrichment period of 8 weeks. In total, 15 of 24 composts tested positive for Legionella species, a higher level of contamination than previously seen in Europe. Two isolates of Legionella pneumophila were identified, and Legionella longbeachae serogroup 1 was found to be one of the most commonly isolated species. L. longbeachae infection would not be detected by routine Legionella urinary antigen assay, so such testing should not be used as the sole diagnostic technique in atypical pneumonia cases, particularly where there is an association with compost use. The occurrence of Legionella in over half of the samples tested indicates that compost could pose a public health risk. The addition of general hygiene warnings to compost packages may be beneficial in protecting public health.

Identification of vaccine antigens using integrated proteomic analyses of surface immunogens from serogroup B Neisseria meningitidis

Meningococcal surface proteins capable of evoking a protective immune response are candidates for inclusion in protein-based vaccines against serogroup B Neisseria meningitidis (NmB). In this study, a 2-dimensional (2-D) gel-based platform integrating surface and immune-proteomics was developed to characterize NmB surface protein antigens. The surface proteome was analyzed by differential 2-D gel electrophoresis following treatment of live bacteria with proteinase K. Alongside, proteins recognized by immune sera from mice challenged with live meningococci were detected using 2-D immunoblots. In combination, seventeen proteins were identified including the well documented antigens PorA, OpcA and factor H-binding protein, previously reported potential antigens and novel potential immunogens. Results were validated for the macrophage infectivity potentiator (MIP), a recently proposed NmB vaccine candidate. MIP-specific antisera bound to meningococci in whole-cell ELISA and facilitated opsonophagocytosis and deposition of complement factors on the surface of meningococcal isolates of different serosubtypes. Cleavage by proteinase K was confirmed in western blots and shown to occur in a fraction of the MIP expressed by meningococci suggesting transient or limited surface exposure. These observations add knowledge for the development of a protein NmB vaccine. The proteomic workflow presented here may be used for the discovery of vaccine candidates against other pathogens. This study presents an integrated proteomic strategy to identify proteins from N. meningitidis with desirable properties (i.e. surface exposure and immunogenicity) for inclusion in subunit vaccines against bacterial meningitis. The effectiveness of the method was demonstrated by the identification of some of the major meningococcal vaccine antigens. Information was also obtained about novel potential immunogens as well as the recently described potential antigen macrophage infectivity potentiator which can be useful for its consideration as a vaccine candidate. Additionally, the proteomic strategy presented in this study provides a generic 2-D gel-based platform for the discovery of vaccine candidates against other bacterial infections.

Proteomic regulation during Legionella pneumophila biofilm development: decrease of virulence factors and enhancement of response to oxidative stress.

Legionella pneumophila (L. pneumophila) is a Gram-negative bacterium, which can be found worldwide in aquatic environments. It tends to persist because it is often protected within biofilms or amoebae. L. pneumophila biofilms have a major impact on water systems, making the understanding of the bacterial physiological adaptation in biofilms a fundamental step towards their eradication. In this study, we report for the first time the influence of the biofilm mode of growth on the proteome of L. pneumophila. We compared the protein patterns of microorganisms grown as suspensions, cultured as colonies on agar plates or recovered with biofilms formed on stainless steel coupons. Statistical analyses of the protein expression data set confirmed the biofilm phenotype specificity which had been previously observed. It also identified dozens of proteins whose abundance was modified in biofilms. Proteins corresponding to virulence factors (macrophage infectivity potentiator protein, secreted proteases) were largely repressed in adherent cells. In contrast, a peptidoglycan-associated lipoprotein (Lpg2043) and a peroxynitrite reductase (Lpg2965) were accumulated by biofilm cells. Remarkably, hypothetical proteins, that appear to be unique to the Legionella genus (Lpg0563, Lpg1111 and Lpg1809), were over-expressed by sessile bacteria.

Detection of Legionella spp. and Legionella pneumophila in water samples of Spain by specific real-time PCR

Legionella pneumophila is the primary cause of the legionellosis diseases (90%) (Yu et al. in J Infect Dis 186:127-128, 2002; Doleans et al. in J Clin Microbiol 42:458-460, 2004; Den Boer et al. in Clin Microbiol Infect 14:459-466, 2008). In this study, methodologies based on molecular biology were developed in order to provide a quick diagnosis of the bacterial presence in water samples of Spain. Multiplex real-time polymerase chain reaction assays were realized to target the 16S rRNA and macrophage infectivity potentiator (mip) genes of, respectively, Legionella spp. and L. pneumophila including in the design of an internal control. The results obtained by the culture and the gene amplification methods agreed in 94.44% for the 16S rRNA gene, and a concordance of 66.67% of the cases was obtained for the mip gene.

Profiling Antibody Responses to Infections by Chlamydia abortus Enables Identification of Potential Virulence Factors and Candidates for Serodiagnosis

Enzootic abortion of ewes (EAE) due to infection with the obligate intracellular pathogen Chlamydia (C.) abortus is an important zoonosis leading to considerable economic loss to agriculture worldwide. The pathogen can be transmitted to humans and may lead to serious infection in pregnant women. Knowledge about epidemiology, clinical course and transmission to humans is hampered by the lack of reliable diagnostic tools. Immunoreactive proteins, which are expressed in infected animals and humans, may serve as novel candidates for diagnostic marker proteins and represent putative virulence factors. In order to broaden the spectrum of immunogenic C. abortus proteins we applied 2D immunoblot analysis and screening of an expression library using human and animal sera. We have identified 48 immunoreactive proteins representing potential diagnostic markers and also putative virulence factors, such as CAB080 (homologue of the “macrophage infectivity potentiator”, MIP), CAB167 (homologue of the “translocated actin recruitment protein”, TARP), CAB712 (homologue of the “chlamydial protease-like activity factor”, CPAF), CAB776 (homologue of the “Polymorphic membrane protein D”, PmpD), and the “hypothetical proteins” CAB063, CAB408 and CAB821, which are predicted to be type III secreted. We selected two putative virulence factors for further characterization, i.e. CAB080 (cMIP) and CAB063, and studied their expression profiles at transcript and protein levels. Analysis of the subcellular localization of both proteins throughout the developmental cycle revealed CAB063 being the first C. abortus protein shown to be translocated to the host cell nucleus.

VBNC Legionella pneumophila cells are still able to produce virulence proteins

Legionella pneumophila is the agent responsible for legionellosis. Numerous bacteria, including L. pneumophila, can enter into a viable but not culturable (VBNC) state under unfavorable environmental conditions. In this state, cells are unable to form colonies on standard medium but are still alive. Here we show that VBNC L. pneumophila cells, obtained by monochloramine treatment, were still able to synthesize proteins, some of which are involved in virulence. Protein synthesis was measured using 35S-labeling and the proteomes of VBNC and culturable cells then compared. This analysis allowed the identification of nine proteins that were accumulated in the VBNC state. Among them, four were involved in virulence, i.e., the macrophage infectivity potentiator protein, the hypothetical protein lpl2247, the ClpP protease proteolytic subunit and the 27 kDa outer membrane protein. Others, i.e., the enoyl reductase, the electron transfer flavoprotein (alpha and beta subunits), the 50S ribosomal proteins (L1 and L25) are involved in metabolic and energy production pathways. However, resuscitation experiments performed with Acanthamoeba castellanii failed, suggesting that the accumulation of virulence factors by VBNC cells is not sufficient to maintain their virulence.

Backbone and side-chain 1H, 13C and 15N assignments of the PPIase domain of macrophage infectivity potentiator (Mip) protein from Coxiella burnetii

Coxiella burnetii is an obligate intracellular gram-negative bacterium uniquely evolved to thrive in the inhospitable phagolysosome of macrophage. C. burnetii causes Q fever in humans and animals, which is emerging as a global public health concern. It is highly infectious and designated as a category B biowarfare agent because of its ubiquitous nature, abundant natural reservoirs, high resistance to environmental conditions, ease of transmission and low infectious dose. The lack of knowledge and awareness of C. burnetii leads to under-reporting and under-diagnosing of Q fever cases. Therefore, further understanding of the interactions between the infected host and the bacteria is necessary. C. burnetii macrophage infectivity potentiator (cb-Mip) is a secreted protein of 230 amino acids involving in intracellular survival of the pathogen. cb-Mip belongs to the family of FK506 binding protein, which possesses peptidyl-prolyl cis/trans isomerase (PPIase) activity. Besides acting as a PPIase, Mip protein homolog has been identified as virulence factor of many intracellular pathogenic microorganisms. In the present study, we report the near complete resonance assignments of the PPIase domain-containing region of Mip protein of C. burnetii. Secondary structure prediction based on chemical shift index analysis indicates that the protein adopts a predominately beta-strand structure, which is consistent with the crystal structure of homologous Mip protein in Legionella pneumophila.

Pathogen-free screening of bacteria-specific hybridomas for selecting high-quality monoclonal antibodies against pathogen bacteria as illustrated for Legionella pneumophila

Antibodies are potent biological tools increasingly used as detection, diagnostic and therapeutic reagents. Many technological advances have optimized and facilitated production and screening of monoclonal antibodies. We report here an original method to screen for antibodies targeting biosafety level 2 or 3 pathogens without the fastidious handling inherent to pathogen use. A double ELISA screening was performed using as coated antigen transformed Escherichia coli expressing at its surface a protein specific to the pathogenic bacteria versus control untransformed E. coli. This method was applied to Legionella, using the surface-exposed Mip protein (macrophage infectivity potentiator). This screening proved to be an excellent means of selecting mAbs that bind Legionella pneumophila 1 surface-exposed Mip protein. This method also appears more biologically relevant than screening using the recombinant Mip protein alone and less tedious than a test performed directly on Legionella bacteria. We obtained 21 mAbs that bind strongly to L. pneumophila serogroups 1 to 13, and we validated their use in a rapid ELISA (performed in 4.5h) and an immunochromatographic test (20min).

Induction of protective immunity against Chlamydia muridarum intravaginal infection with the chlamydial immunodominant antigen macrophage infectivity potentiator

We previously reported that 5 Chlamydia muridarum antigens reacted with antisera from >90% mice urogenitally infected with C. muridarum and they are TC0660 (ABC transporter or ArtJ), TC0727 (outer membrane complex protein B or OmcB), TC0828 (macrophage infectivity potentiator or MIP), TC0726 (inclusion membrane protein or Inc) & TC0268 (hypothetical protein or HP). The orthologs of these antigens in Chlamydia trachomatis were also highly reactive with antisera from women urogenitally infected with C. trachomatis. In the current study, we evaluated these C. muridarum antigens for their ability to induce protection against a C. muridarum intravaginal challenge infection in mice. We found that only MIP induced the most pronounced protection against C. muridarum infection. The protection correlated well with robust C. muridarum MIP-specific antibody and Th1-dominant T cell responses. The MIP-immunized mice displayed significantly reduced live organism shedding from the lower genital tract and highly attenuated inflammatory pathologies in the upper genital tissues. These results demonstrate that MIP, an immunodominant antigen identified by both human and mouse antisera, may be considered a component of a multi-subunit chlamydial vaccine for inducing protective immunity.