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Michael A Apicella - One of the best experts on this subject based on the ideXlab platform.
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Nontypeable Haemophilus influenzae lipooligosaccharide expresses a terminal ketodeoxyoctanoate in vivo, which can be used as a target for bactericidal antibody
mBio, 2018Co-Authors: Michael A Apicella, Margaret Ketterer, Deborah M.b. Post, Freda E.c. Jen, Christopher J. Day, Jeremy Coffin, Michael P JenningsAbstract:Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in individuals of all ages. The lipooligosaccharide (LOS) of NTHi has evolved a complex structure that can be attributed to a multiplicity of glycosyltransferases, the random switching of glycosyltransferase gene expression via phase variation, and the complex structure of its core region with multiple glycoform branch points. This article adds to that complexity by describing a multifunctional enzyme (LsgB) which optimally functions when the species is grown on a solid surface and which can add either a ketodeoxyoctanoate (KDO) or an N -acetylneuramic acid (Neu5Ac) moiety to a terminal N- acetyllactosamine structure of LOS. Our studies show that expression of lsgB is reduced four- to sixfold when NTHi is grown in broth. The substrate that the enzyme utilizes is dependent upon the concentration of free Neu5Ac (between 1 and 10 µg/ml) in the environment. In environments in which Neu5Ac is below that level, the enzyme utilizes endogenous CMP-KDO as the substrate. Our studies show that during in vivo growth in an NTHi biofilm, the KDO moiety is expressed by the organism. Monoclonal antibody 6E4, which binds KDO, is bactericidal for NTHi strains that express the KDO epitope at high levels. In a survey of 33 NTHi strains isolated from healthy and diseased individuals, the antibody was bactericidal (>90% kill) for 12 strains (36%). These studies open up the possibility of using a KDO-based glycoconjugate vaccine as part of a multicomponent vaccine against NTHi. IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in middle ear infections in children, sinusitis in adults, and acute bronchitis in individuals with chronic obstructive lung disease. The organism is very well adapted to the human host environment, and this has hindered successful development of an effective vaccine. In this article, we describe a mechanism by which the bacteria decorates its surface lipooligosaccharide with a sugar unique to Gram-negative bacteria, ketodeoxyoctanoate (KDO). This sugar decoration is present during active infection and we have shown that an antibody directed against this sugar can result in killing of the organism. These data demonstrate that the lipooligosaccharide ketodeoxyoctanoate epitope may be a novel NTHi - specific candidate vaccine antigen.
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Effect of lipooligosaccharide mutations of Haemophilus influenzae on the Middle and Inner Ears
International journal of pediatric otorhinolaryngology, 2009Co-Authors: Patricia A. Schachern, Michael A Apicella, Vladimir Tsuprun, Beinan Wang, Sebahattin Cureoglu, Michael M. Paparella, Steven K. JuhnAbstract:Objective The purpose of this study was to determine the virulence of nontypeable Haemophilus influenzae 2019 (NTHi 2019) and its two lipooligosaccharide (LOS) mutant strains, B29 (gene htrB) and DK1 (gene rfaD), and compare their effect on the middle ear, round window membrane, and inner ear.
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Novel Sialic Acid Transporter of Haemophilus influenzae
Infection and Immunity, 2005Co-Authors: Simon Allen, Anthony Zaleski, Jason W. Johnston, Bradford W. Gibson, Michael A ApicellaAbstract:Nontypeable Haemophilus influenzae is an opportunistic pathogen and a common cause of otitis media in children and of chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The Lipooligosaccharides, a major component of the outer membrane of H. influenzae, play an important role in microbial virulence and pathogenicity. N-Acetylneuraminic acid (sialic acid) can be incorporated into the Lipooligosaccharides as a terminal nonreducing sugar. Although much of the pathway of sialic acid incorporation into Lipooligosaccharides is understood, the transporter responsible for N-acetylneuraminic acid uptake in H. influenzae has yet to be characterized. In this paper we demonstrate that this transporter is a novel sugar transporter of the tripartite ATP-independent periplasmic transporter family. In the absence of this transporter, H. influenzae cannot incorporate sialic acid into its Lipooligosaccharides, making the organism unable to survive when exposed to human serum and causing reduced viability in biofilm growth.
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Susceptibility of Nontypeable Haemophilus influenzae to Human β-Defensins Is Influenced by Lipooligosaccharide Acylation
Infection and immunity, 2002Co-Authors: Timothy D. Starner, Michael A Apicella, W. Edward Swords, Paul B. MccrayAbstract:Nontypeable Haemophilus influenzae (NTHI) lipooligosaccharide htrB mutants exhibited greater than 45-fold-increased sensitivity to human β-defensin 2 (HBD-2) compared to the wild type. Complementation by htrB in trans to acylation competence reversed this increased sensitivity. In contrast, NTHI was more susceptible to HBD-3 and showed no changes in sensitivity as a result of lipooligosaccharide mutations in oligosaccharide and lipid A biosynthesis genes.
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Human immune response to monoclonal antibody — defined epitopes of Neisseria gonorrhoeae Lipooligosaccharides
Gonococci and Meningococci, 1998Co-Authors: Robert E. Mandrell, Michael A Apicella, Peter A. Rice, John W. Boslego, R. Chung, J. Mcleod GriffissAbstract:Strains of Neisseria gonorrhoeae isolated from patients with local (LI) or disseminated (DGI) infections were screened with a panel of ten monoclonal antibodies (MAb) specific for components of Neisseria Lipooligosaccharides (LOS). Homologous pre-infection, acute and convalescent sera were tested in a solid phase radioimmunoassay as competitors of binding of relevant MAb’s to epitopes on the LOS. Sixteen of 35 (46%) total epitope reactions were competed by DGI convalescent sera compared to 2 of 28 (7%) for LI convalescent sera. Competitive binding by DGI sera reflected higher levels of total LOS antibody in DGI sera compared to LI sera.
Bradford W. Gibson - One of the best experts on this subject based on the ideXlab platform.
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Stable isotope metabolic labeling of Neisseria meningitidis lipooligosaccharide.
Journal of endotoxin research, 2006Co-Authors: Deborah M.b. Post, Jerrold Weiss, Desheng Zhang, Bradford W. GibsonAbstract:The lipooligosaccharide (LOS) of a Neisseria meningitidis acetate auxotroph was metabolically labeled with either [2- 13 C]-sodium acetate or [1,2- 13 C 2 ]-sodium acetate. In this study, we demonstrated that this label was efficiently incorporated into both the lipid A acyl moieties and the two N-acetylglucosamines present in the oligosaccharide branch of the LOS. The development of this efficient labeling protocol should prove useful in future structural studies analyzing the interactions between LOS and host proteins.
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Novel Sialic Acid Transporter of Haemophilus influenzae
Infection and Immunity, 2005Co-Authors: Simon Allen, Anthony Zaleski, Jason W. Johnston, Bradford W. Gibson, Michael A ApicellaAbstract:Nontypeable Haemophilus influenzae is an opportunistic pathogen and a common cause of otitis media in children and of chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The Lipooligosaccharides, a major component of the outer membrane of H. influenzae, play an important role in microbial virulence and pathogenicity. N-Acetylneuraminic acid (sialic acid) can be incorporated into the Lipooligosaccharides as a terminal nonreducing sugar. Although much of the pathway of sialic acid incorporation into Lipooligosaccharides is understood, the transporter responsible for N-acetylneuraminic acid uptake in H. influenzae has yet to be characterized. In this paper we demonstrate that this transporter is a novel sugar transporter of the tripartite ATP-independent periplasmic transporter family. In the absence of this transporter, H. influenzae cannot incorporate sialic acid into its Lipooligosaccharides, making the organism unable to survive when exposed to human serum and causing reduced viability in biofilm growth.
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Characterization of Two Transposon Mutants from Haemophilus influenzae Type b with Altered Lipooligosaccharide Biosynthesis
Biochemistry, 1996Co-Authors: Nancy J. Phillips, Michael A Apicella, Robert E. Mclaughlin, Theresa J. Miller, Bradford W. GibsonAbstract:Two isogenic mutants of Haemophilus influenzae type b (Hib) strain A2 were prepared by random m-Tn3(Cm) insertions into the 7.4-kb lsg (lipooligosaccharide synthesis genes) region of Hib DNA, which consists of seven complete and one partial open reading frames (orfs). Compared to the parent A2 strain which produces a complex mixture of Lipooligosaccharides (LOS), the mutant strains 281.25 and 276.4 produced only a few LOS species. The precise locations of transposon insertions into the lsg loci of these mutants were determined (base 3546 in orf 4 for strain 281.25 and base 4402 in orf 5 for strain 276.4), and the effects of these mutations on LOS biosynthesis and epitope expression were evaluated. When the O-deacylated LOS were analyzed by mass spectrometry, both strains contained major LOS species of Mr 2601, 2439, and 2277, which consisted of a common heptose trisaccharide core structure [Hep3(PEA)Kdo(P)-lipid A, where Hep is l-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-octulosonic acid, and PEA is...
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The Lipooligosaccharides of Haemophilus ducreyi are highly sialylated.
Journal of bacteriology, 1996Co-Authors: William Melaugh, Anthony A. Campagnari, Bradford W. GibsonAbstract:The major Lipooligosaccharides of the sexually transmitted pathogen Haemophilus ducreyi 35000 have been previously found to terminate in N-acetyllactosamine and sialyl-N-acetyllactosamine, Neu5Ac alpha 2-->3Gal beta 1-->4GlcNAc (W. Melaugh, N. J. Phillips, A. A. Campagnari, M. V. Tullius, and B. W. Gibson, Biochemistry 33: 13070-13078, 1994). In this study, mass spectrometry and composition analyses have shown that the Lipooligosaccharides from three other H. ducreyi strains also contain N-acetyllactosamine and are highly sialylated (approximately 30 to 50%), although one African strain was found to contain neither of these structural features.
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The Lipooligosaccharides of pathogenic Gram-negative bacteria
Critical reviews in microbiology, 1996Co-Authors: Andrew Preston, Bradford W. Gibson, Robert E. Mandrell, Michael A ApicellaAbstract:Lipooligosaccharides (LOSs) are the major glycolipids expressed on mucosal Gram-negative bacteria, including members of the genera Neisseria, Haemophilus, Bordetella, and Branhamella. They can also be expressed on some enteric bacteria such as Campylobacter jejuni and Campylobacter coli strains. LOS is analogous to the lipopolysaccharide (LPS) found in other Gram-negative families. LOSs share similar lipid A structures with an identical array of functional activities as LPSs. LOSs lack O-antigen units with the LOS oligosaccharide structures limited to 10 saccharide units. The LOS species of pathogenic Neisseria can play a major role in pathogenesis through enhancing the resistance of the organism to killing by normal human serum. Other distinguishing characteristics of LOS are the structural and antigenic similarity of some LOS species to human glycolipids and the potential for certain LOSs to be modified in vivo by host substances or secretions. These modifications of LOS in different environments of the host result in synthesis of new LOS structures that probably benefit the survival of the pathogen. The LOS of N. gonorrhoeae can act as a ligand of human receptors, promoting invasion of host cells. It is becoming clearer that LOSs are crucial factors in the pathogenesis of bacteria that express them.
Christiane Bouchier - One of the best experts on this subject based on the ideXlab platform.
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pks5-recombination-mediated surface remodelling in Mycobacterium tuberculosis emergence
Nature Microbiology, 2016Co-Authors: Eva C. Boritsch, Wladimir Malaga, Alexandre Pawlik, Wafa Frigui, Alessandro Cascioferro, Gilles Etienne, Françoise Laval, Fabien Le Chevalier, Mickael Orgeur, Christiane BouchierAbstract:pks5 -recombination-mediated cell surface remodelling increased virulence of Mycobacterium canettii , driving evolution from a putative generalist mycobacteria towards a professional pathogen of mammalian hosts. Mycobacterium tuberculosis is a major, globally spread, aerosol-transmitted human pathogen, thought to have evolved by clonal expansion from a Mycobacterium canettii -like progenitor. In contrast, extant M. canettii strains are rare, genetically diverse, and geographically restricted mycobacteria of only marginal epidemiological importance. Here, we show that the contrasting evolutionary success of these two groups is linked to loss of lipooligosaccharide biosynthesis and subsequent morphotype changes. Spontaneous smooth-to-rough M. canettii variants were found to be mutated in the polyketide-synthase-encoding pks5 locus and deficient in lipooligosaccharide synthesis, a phenotype restored by complementation. Importantly, these rough variants showed an altered host–pathogen interaction and increased virulence in cellular- and animal-infection models. In one variant, lipooligosaccharide deficiency occurred via homologous recombination between two pks5 genes and removal of the intervening acyltransferase-encoding gene. The resulting single pks5 configuration is similar to that fixed in M. tuberculosis , which is known to lack Lipooligosaccharides. Our results suggest that pks5 -recombination-mediated bacterial surface remodelling increased virulence, driving evolution from putative generalist mycobacteria towards professional pathogens of mammalian hosts.
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Pks5-recombination-mediated surface remodelling in Mycobacterium tuberculosis emergence
Nature Microbiology, 2016Co-Authors: Eva C. Boritsch, Wladimir Malaga, Alexandre Pawlik, Wafa Frigui, Alessandro Cascioferro, Gilles Etienne, Françoise Laval, Fabien Le Chevalier, Mickael Orgeur, Christiane BouchierAbstract:Mycobacterium tuberculosis is a major, globally spread, aerosol-transmitted human pathogen, thought to have evolved by clonal expansion from a Mycobacterium canettii-like progenitor. In contrast, extant M. canettii strains are rare, genetically diverse, and geographically restricted mycobacteria of only marginal epidemiological importance. Here, we show that the contrasting evolutionary success of these two groups is linked to loss of lipooligosaccharide biosynthesis and subsequent morphotype changes. Spontaneous smooth-to-rough M. canettii variants were found to be mutated in the polyketide-synthase-encoding pks5 locus and deficient in lipooligosaccharide synthesis, a phenotype restored by complementation. Importantly, these rough variants showed an altered host–pathogen interaction and increased virulence in cellular- and animal-infection models. In one variant, lipooligosaccharide deficiency occurred via homologous recombination between two pks5 genes and removal of the intervening acyltransferase-encoding gene. The resulting single pks5 configuration is similar to that fixed in M. tuberculosis, which is known to lack Lipooligosaccharides. Our results suggest that pks5-recombination-mediated bacterial surface remodelling increased virulence, driving evolution from putative generalist mycobacteria towards professional pathogens of mammalian hosts. Tuberculosis is a major human infectious disease. Although many aspects of the disease-causing potential of its aetiological agent M. tuberculosis are known1, our understanding of the molecular events that favoured its evolutionary success as one of the most widely distributed human pathogens remains scant. New insights into this question are important for uncovering the mechanisms of pathogenesis and new drug targets2. Strains of the closely related and phylogenetically early branching M. canettii, also named ‘smooth tubercle bacilli’ (STB)3, are powerful resources to investigate the evolution of M. tuberculosis and the M. tuberculosis complex (MTBC)3. The first strain of M. canettii was isolated by Georges Canetti in 1969, and since then, fewer than 100 isolates have been described, most of which have been isolated from tuberculosis patients with a connection to the Horn of Africa4,5,6,7. Despite their geographic restriction, M. canettii strains show much greater genetic variability and are less virulent/persistent than M. tuberculosis. Genome comparisons suggest that M. tuberculosis evolved by clonal expansion from a pool of M. canettii-like tubercle bacilli through the gain of virulence and persistence mechanisms3,8,9. Although some genomic differences have been found to be specific for a single M. canettii strain, apparently due to isolated horizontal gene transfer (for example, the mce5 operon3 or the eptABCD operon10, present exclusively in strain STB-J)3, others are conserved throughout all M. canettii strains as a result of phylogenetic ancestry (for example, cobF, which is present in M. canettii and deleted from the MTBC)3. Here, we investigate phenotypic differences between M. canettii and M. tuberculosis and focus on the unique, conserved, smooth (S) colony morphotype of M. canettii that contrasts with the rough (R) morphotype of MTBC members. Previously, S morphotypes of non-tuberculous mycobacterial species such as Mycobacterium avium11, Mycobacterium abscessus12, Mycobacterium kansasii13 or Mycobacterium marinum14 have been found to be less virulent than R morphotypes, raising the question of whether the highly conserved M. tuberculosis R morphotype might have been the result of evolutionary selection, based on host–pathogen interactions favouring a more virulent or persistent phenotype. In other mycobacterial species, S/R variation is often attributed to different kinds of cell surface glycolipid, such as glycopeptidolipid (GPL) for M. avium15 and M. abscessus16,17 or lipooligosaccharide (LOS) for M. kansasii13 and M. marinum14,18,19. Insights from earlier studies of M. canettii have remained abstruse, as no specific lipid exclusively present in the S morphotype has been identified20, nor has the genetic basis for morphotype variation been determined4. Building on recent data from several M. canettii genomes3, we studied smooth and spontaneously converted R variants of two different M. canettii strains, STB-K (CIPT 140070010) and STB-I (CIPT 140070007)3, hereafter termed KS/R and IS/R, respectively. We used whole genome sequencing (WGS) and identified differences in the genes of the pks5 locus, which in Mycobacterium smegmatis, M. marinum or M. kansasii are implicated in LOS biosynthesis14,21,22,23. In this Article, we uncover the mechanisms underlying the S-to-R morphology change of tubercle bacilli and explore the biological consequences with emphasis on the patho-evolution of M. tuberculosis.
Robert E. Mandrell - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Lipooligosaccharide-Biosynthetic Loci of Campylobacter jejuni Reveals New Lipooligosaccharide Classes: Evidence of Mosaic Organizations
Journal of bacteriology, 2008Co-Authors: Craig T Parker, Nobuhiro Yuki, Michel Gilbert, Hubert P. Endtz, Robert E. MandrellAbstract:The lipooligosaccharide (LOS) biosynthesis region is one of the more variable genomic regions between strains of Campylobacter jejuni. Indeed, eight classes of LOS biosynthesis loci have been established previously based on gene content and organization. In this study, we characterize additional classes of LOS biosynthesis loci and analyze various mechanisms that result in changes to LOS structures. To gain further insights into the genomic diversity of C. jejuni LOS biosynthesis region, we sequenced the LOS biosynthesis loci of 15 strains that possessed gene content that was distinct from the eight classes. This analysis identified 11 new classes of LOS loci that exhibited examples of deletions and insertions of genes and cassettes of genes found in other LOS classes or capsular biosynthesis loci leading to mosaic LOS loci. The sequence analysis also revealed both missense mutations leading to "allelic" glycosyltransferases and phase-variable and non-phase-variable gene inactivation by the deletion or insertion of bases. Specifically, we demonstrated that gene inactivation is an important mechanism for altering the LOS structures of strains possessing the same class of LOS biosynthesis locus. Together, these observations suggest that LOS biosynthesis region is a hotspot for genetic exchange and variability, often leading to changes in the LOS produced.
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major structural differences and novel potential virulence mechanisms from the genomes of multiple campylobacter species
PLOS Biology, 2005Co-Authors: Derrick E Fouts, Robert E. Mandrell, Emmanuel F Mongodin, William G Miller, David A Rasko, Jacques Ravel, Lauren M Brinkac, Robert T Deboy, Craig T Parker, Sean C DaughertyAbstract:Sequencing and comparative genome analysis of four strains of Campylobacter including C. lari RM2100, C. upsaliensis RM3195, and C. coli RM2228 has revealed major structural differences that are associated with the insertion of phage- and plasmid-like genomic islands, as well as major variations in the lipooligosaccharide complex. Poly G tracts are longer, are greater in number, and show greater variability in C. upsaliensis than in the other species. Many genes involved in host colonization, including racR/S, cadF, cdt, ciaB, and flagellin genes, are conserved across the species, but variations that appear to be species specific are evident for a lipooligosaccharide locus, a capsular (extracellular) polysaccharide locus, and a novel Campylobacter putative licABCD virulence locus. The strains also vary in their metabolic profiles, as well as their resistance profiles to a range of antibiotics. It is evident that the newly identified hypothetical and conserved hypothetical proteins, as well as uncharacterized two-component regulatory systems and membrane proteins, may hold additional significant information on the major differences in virulence among the species, as well as the specificity of the strains for particular hosts.
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Human immune response to monoclonal antibody — defined epitopes of Neisseria gonorrhoeae Lipooligosaccharides
Gonococci and Meningococci, 1998Co-Authors: Robert E. Mandrell, Michael A Apicella, Peter A. Rice, John W. Boslego, R. Chung, J. Mcleod GriffissAbstract:Strains of Neisseria gonorrhoeae isolated from patients with local (LI) or disseminated (DGI) infections were screened with a panel of ten monoclonal antibodies (MAb) specific for components of Neisseria Lipooligosaccharides (LOS). Homologous pre-infection, acute and convalescent sera were tested in a solid phase radioimmunoassay as competitors of binding of relevant MAb’s to epitopes on the LOS. Sixteen of 35 (46%) total epitope reactions were competed by DGI convalescent sera compared to 2 of 28 (7%) for LI convalescent sera. Competitive binding by DGI sera reflected higher levels of total LOS antibody in DGI sera compared to LI sera.
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human immune response to monoclonal antibody defined epitopes of neisseria gonorrhoeae Lipooligosaccharides
1998Co-Authors: Robert E. Mandrell, Michael A Apicella, Peter A. Rice, John W. Boslego, R. Chung, Mcleod J GriffissAbstract:Strains of Neisseria gonorrhoeae isolated from patients with local (LI) or disseminated (DGI) infections were screened with a panel of ten monoclonal antibodies (MAb) specific for components of Neisseria Lipooligosaccharides (LOS). Homologous pre-infection, acute and convalescent sera were tested in a solid phase radioimmunoassay as competitors of binding of relevant MAb’s to epitopes on the LOS. Sixteen of 35 (46%) total epitope reactions were competed by DGI convalescent sera compared to 2 of 28 (7%) for LI convalescent sera. Competitive binding by DGI sera reflected higher levels of total LOS antibody in DGI sera compared to LI sera.
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Immunogenicity of Neisseria gonorrhoeae Lipooligosaccharide Epitope 2C7, Widely Expressed In Vivo with No Immunochemical Similarity to Human Glycosphingolipids
The Journal of infectious diseases, 1996Co-Authors: Sunita Gulati, Daniel P. Mcquillen, Robert E. Mandrell, Darshana B. Jani, Peter A. RiceAbstract:Natural infection with Neisseria gonorrhoeae may elicit a substantial antibody response directed against gonococcal lipooligosaccharide. Monoclonal antibody (MAb) 2C7 recognized a gonococcal lipooligosaccharide epitope, identified the epitope directly in 94% of 68 consecutive culture-positive genital secretions, and recognized 95% of 101 randomly chosen fresh (second-passage) gonococcal isolates. The epitope was stably maintained after multiple in vitro passages and did not compete with any of the known cross-reactive human glycosphingolipid structures. MAb 2C7 mediated in vitro killing and phagocytosis by human polymorphonuclear leukocytes of 1 serum-sensitive (sialylated or not) and 1 stably serum-resistant gonococcal isolate that expressed the epitope. Gonococcal endometritis and disseminated infection elicited increases (6.5-fold IgM, 4.4-fold IgG; 18-fold IgM, 17-fold IgG, respectively) in anti-2C7 epitope antibody. Immunization with a gonococcal outer membrane vaccine elicited a mean 44.5-fold increase in IgG anti-2C7 epitope antibody in 20 of 28 subjects. The epitope identified by MAb 2C7 may represent an excellent target for a potentially protective gonococcal vaccine candidate.
Eva C. Boritsch - One of the best experts on this subject based on the ideXlab platform.
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pks5-recombination-mediated surface remodelling in Mycobacterium tuberculosis emergence
Nature Microbiology, 2016Co-Authors: Eva C. Boritsch, Wladimir Malaga, Alexandre Pawlik, Wafa Frigui, Alessandro Cascioferro, Gilles Etienne, Françoise Laval, Fabien Le Chevalier, Mickael Orgeur, Christiane BouchierAbstract:pks5 -recombination-mediated cell surface remodelling increased virulence of Mycobacterium canettii , driving evolution from a putative generalist mycobacteria towards a professional pathogen of mammalian hosts. Mycobacterium tuberculosis is a major, globally spread, aerosol-transmitted human pathogen, thought to have evolved by clonal expansion from a Mycobacterium canettii -like progenitor. In contrast, extant M. canettii strains are rare, genetically diverse, and geographically restricted mycobacteria of only marginal epidemiological importance. Here, we show that the contrasting evolutionary success of these two groups is linked to loss of lipooligosaccharide biosynthesis and subsequent morphotype changes. Spontaneous smooth-to-rough M. canettii variants were found to be mutated in the polyketide-synthase-encoding pks5 locus and deficient in lipooligosaccharide synthesis, a phenotype restored by complementation. Importantly, these rough variants showed an altered host–pathogen interaction and increased virulence in cellular- and animal-infection models. In one variant, lipooligosaccharide deficiency occurred via homologous recombination between two pks5 genes and removal of the intervening acyltransferase-encoding gene. The resulting single pks5 configuration is similar to that fixed in M. tuberculosis , which is known to lack Lipooligosaccharides. Our results suggest that pks5 -recombination-mediated bacterial surface remodelling increased virulence, driving evolution from putative generalist mycobacteria towards professional pathogens of mammalian hosts.
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Pks5-recombination-mediated surface remodelling in Mycobacterium tuberculosis emergence
Nature Microbiology, 2016Co-Authors: Eva C. Boritsch, Wladimir Malaga, Alexandre Pawlik, Wafa Frigui, Alessandro Cascioferro, Gilles Etienne, Françoise Laval, Fabien Le Chevalier, Mickael Orgeur, Christiane BouchierAbstract:Mycobacterium tuberculosis is a major, globally spread, aerosol-transmitted human pathogen, thought to have evolved by clonal expansion from a Mycobacterium canettii-like progenitor. In contrast, extant M. canettii strains are rare, genetically diverse, and geographically restricted mycobacteria of only marginal epidemiological importance. Here, we show that the contrasting evolutionary success of these two groups is linked to loss of lipooligosaccharide biosynthesis and subsequent morphotype changes. Spontaneous smooth-to-rough M. canettii variants were found to be mutated in the polyketide-synthase-encoding pks5 locus and deficient in lipooligosaccharide synthesis, a phenotype restored by complementation. Importantly, these rough variants showed an altered host–pathogen interaction and increased virulence in cellular- and animal-infection models. In one variant, lipooligosaccharide deficiency occurred via homologous recombination between two pks5 genes and removal of the intervening acyltransferase-encoding gene. The resulting single pks5 configuration is similar to that fixed in M. tuberculosis, which is known to lack Lipooligosaccharides. Our results suggest that pks5-recombination-mediated bacterial surface remodelling increased virulence, driving evolution from putative generalist mycobacteria towards professional pathogens of mammalian hosts. Tuberculosis is a major human infectious disease. Although many aspects of the disease-causing potential of its aetiological agent M. tuberculosis are known1, our understanding of the molecular events that favoured its evolutionary success as one of the most widely distributed human pathogens remains scant. New insights into this question are important for uncovering the mechanisms of pathogenesis and new drug targets2. Strains of the closely related and phylogenetically early branching M. canettii, also named ‘smooth tubercle bacilli’ (STB)3, are powerful resources to investigate the evolution of M. tuberculosis and the M. tuberculosis complex (MTBC)3. The first strain of M. canettii was isolated by Georges Canetti in 1969, and since then, fewer than 100 isolates have been described, most of which have been isolated from tuberculosis patients with a connection to the Horn of Africa4,5,6,7. Despite their geographic restriction, M. canettii strains show much greater genetic variability and are less virulent/persistent than M. tuberculosis. Genome comparisons suggest that M. tuberculosis evolved by clonal expansion from a pool of M. canettii-like tubercle bacilli through the gain of virulence and persistence mechanisms3,8,9. Although some genomic differences have been found to be specific for a single M. canettii strain, apparently due to isolated horizontal gene transfer (for example, the mce5 operon3 or the eptABCD operon10, present exclusively in strain STB-J)3, others are conserved throughout all M. canettii strains as a result of phylogenetic ancestry (for example, cobF, which is present in M. canettii and deleted from the MTBC)3. Here, we investigate phenotypic differences between M. canettii and M. tuberculosis and focus on the unique, conserved, smooth (S) colony morphotype of M. canettii that contrasts with the rough (R) morphotype of MTBC members. Previously, S morphotypes of non-tuberculous mycobacterial species such as Mycobacterium avium11, Mycobacterium abscessus12, Mycobacterium kansasii13 or Mycobacterium marinum14 have been found to be less virulent than R morphotypes, raising the question of whether the highly conserved M. tuberculosis R morphotype might have been the result of evolutionary selection, based on host–pathogen interactions favouring a more virulent or persistent phenotype. In other mycobacterial species, S/R variation is often attributed to different kinds of cell surface glycolipid, such as glycopeptidolipid (GPL) for M. avium15 and M. abscessus16,17 or lipooligosaccharide (LOS) for M. kansasii13 and M. marinum14,18,19. Insights from earlier studies of M. canettii have remained abstruse, as no specific lipid exclusively present in the S morphotype has been identified20, nor has the genetic basis for morphotype variation been determined4. Building on recent data from several M. canettii genomes3, we studied smooth and spontaneously converted R variants of two different M. canettii strains, STB-K (CIPT 140070010) and STB-I (CIPT 140070007)3, hereafter termed KS/R and IS/R, respectively. We used whole genome sequencing (WGS) and identified differences in the genes of the pks5 locus, which in Mycobacterium smegmatis, M. marinum or M. kansasii are implicated in LOS biosynthesis14,21,22,23. In this Article, we uncover the mechanisms underlying the S-to-R morphology change of tubercle bacilli and explore the biological consequences with emphasis on the patho-evolution of M. tuberculosis.