Yih-Ling Tzeng

A CRISPR/Cas system mediates bacterial innate immune evasion and virulence

CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innate immune response aimed at combating pathogens, CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts.

Epidemiology and pathogenesis of Neisseria meningitidis.

Neisseria meningitidis, an exclusive pathogen of humans, remains the leading worldwide cause of meningitis and fatal sepsis, usually in otherwise healthy individuals. In recent years, significant advances have improved our understanding of the epidemiology and genetic basis of meningococcal disease and led to progress in the development of the next generation of meningococcal vaccines. This review summarizes current knowledge of the human susceptibility to and the epidemiology and molecular pathogenesis of meningococcal disease.

Cationic Antimicrobial Peptide Resistance in Neisseria meningitidis

Cationic antimicrobial peptides (CAMPs) are important components of the innate host defense system against microbial infections and microbial products. However, the human pathogen Neisseria meningitidis is intrinsically highly resistant to CAMPs, such as polymyxin B (PxB) (MIC > or = 512 microg/ml). To ascertain the mechanisms by which meningococci resist PxB, mutants that displayed increased sensitivity (> or =4-fold) to PxB were identified from a library of mariner transposon mutants generated in a meningococcal strain, NMB. Surprisingly, more than half of the initial PxB-sensitive mutants had insertions within the mtrCDE operon, which encodes proteins forming a multidrug efflux pump. Additional PxB-sensitive mariner mutants were identified from a second round of transposon mutagenesis performed in an mtr efflux pump-deficient background. Further, a mutation in lptA, the phosphoethanolamine (PEA) transferase responsible for modification of the lipid A head groups, was identified to cause the highest sensitivity to PxB. Mutations within the mtrD or lptA genes also increased meningococcal susceptibility to two structurally unrelated CAMPs, human LL-37 and protegrin-1. Consistently, PxB neutralized inflammatory responses elicited by the lptA mutant lipooligosaccharide more efficiently than those induced by wild-type lipooligosaccharide. mariner mutants with increased resistance to PxB were also identified in NMB background and found to contain insertions within the pilMNOPQ operon involved in pilin biogenesis. Taken together, these data indicated that meningococci utilize multiple mechanisms including the action of the MtrC-MtrD-MtrE efflux pump and lipid A modification as well as the type IV pilin secretion system to modulate levels of CAMP resistance. The modification of meningococcal lipid A head groups with PEA also prevents neutralization of the biological effects of endotoxin by CAMP.

Neisseria meningitidis Lipooligosaccharide Structure-Dependent Activation of the Macrophage CD14/Toll-Like Receptor 4 Pathway

ABSTRACT Meningococcal lipopoly(oligo)saccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis. Highly purified wild-type meningococcal LOS and LOS from genetically defined mutants of Neisseria meningitidis that contained specific mutations in LOS biosynthesis pathways were used to confirm that meningococcal LOS activation of macrophages was CD14/Toll-like receptor 4 (TLR4)-MD-2 dependent and to elucidate the LOS structural requirement for TLR4 activation. Expression of TLR4 but not TLR2 was required, and antibodies to both TLR4 and CD14 blocked meningococcal LOS activation of macrophages. Meningococcal LOS α or β chain oligosaccharide structure did not influence CD14/TLR4-MD-2 activation. However, meningococcal lipid A, expressed by meningococci with defects in 3-deoxy-d-manno-octulosonic acid (KDO) biosynthesis or transfer, resulted in an ∼10-fold (P < 0.0001) reduction in biologic activity compared to KDO2-containing meningococcal LOS. Removal of KDO2 from LOS by acid hydrolysis also dramatically attenuated cellular responses. Competitive inhibition assays showed similar binding of glycosylated and unglycosylated lipid A to CD14/TLR4-MD-2. A decrease in the number of lipid A phosphate head groups or penta-acylated meningococcal LOS modestly attenuated biologic activity. Meningococcal endotoxin is a potent agonist of the macrophage CD14/TLR4-MD-2 receptor, helping explain the fulminant presentation of meningococcal sepsis and meningitis. KDO2 linked to meningococcal lipid A was structurally required for maximal activation of the human macrophage TLR4 pathway and indicates an important role for KDO-lipid A in endotoxin biologic activity.

Endotoxin of Neisseria meningitidis composed only of intact lipid A: Inactivation of the meningococcal 3-deoxy-D-manno-octulosonic acid transferase

Lipopolysaccharide, lipooligosaccharide (LOS), or endotoxin is important in bacterial survival and the pathogenesis of gram-negative bacteria. A necessary step in endotoxin biosynthesis is 3-deoxy-D-manno-octulosonic acid (Kdo) glycosylation of lipid A, catalyzed by the Kdo transferase KdtA (WaaA). In enteric gram-negative bacteria, this step is essential for survival. A nonpolar kdtA::aphA-3 mutation was created in Neisseria meningitidis via allelic exchange, and the mutant was viable. Detailed structural analysis demonstrated that the endotoxin of the kdtA::aphA-3 mutant was composed of fully acylated lipid A with variable phosphorylation but without Kdo glycosylation. In contrast to what happens in other gram-negative bacteria, tetra-acylated lipid IV(A) did not accumulate. The LOS structure of the kdtA::aphA-3 mutant was restored to the wild-type structure by complementation with kdtA from N. meningitidis or Escherichia coli. The expression of a fully acylated, unglycosylated lipid A indicates that lipid A biosynthesis in N. meningitidis can proceed without the addition of Kdo and that KdtA is not essential for survival of the meningococcus.

Meningococcal disease: changes in epidemiology and prevention

The human bacterial pathogen Neisseria meningitidis remains a serious worldwide health threat, but progress is being made toward the control of meningococcal infections. This review summarizes current knowledge of the global epidemiology and the pathophysiology of meningococcal disease, as well as recent advances in prevention by new vaccines. Meningococcal disease patterns and incidence can vary dramatically, both geographically and over time in populations, influenced by differences in invasive meningococcal capsular serogroups and specific genotypes designated as ST clonal complexes. Serogroup A (ST-5, ST-7), B (ST-41/44, ST-32, ST-18, ST-269, ST-8, ST-35), C (ST-11), Y (ST-23, ST-167), W-135 (ST-11) and X (ST-181) meningococci currently cause almost all invasive disease. Serogroups B, C, and Y are responsible for the majority of cases in Europe, the Americas, and Oceania; serogroup A has been associated with the highest incidence (up to 1000 per 100,000 cases) and large outbreaks of meningococcal disease in sub-Saharan Africa and previously Asia; and serogroups W-135 and X have emerged to cause major disease outbreaks in sub-Saharan Africa. Significant declines in meningococcal disease have occurred in the last decade in many developed countries. In part, the decline is related to the introduction of new meningococcal vaccines. Serogroup C polysaccharide-protein conjugate vaccines were introduced over a decade ago, first in the UK in a mass vaccination campaign, and are now widely used; multivalent meningococcal conjugate vaccines containing serogroups A, C, W-135, and/or Y were first used for adolescents in the US in 2005 and have now expanded indications for infants and young children, and a new serogroup A conjugate vaccine has recently been introduced in sub-Saharan Africa. The effectiveness of these conjugate vaccines has been enhanced by the prevention of person-to-person transmission and herd immunity. In addition, progress has been made in serogroup B-specific vaccines based on conserved proteins and outer membrane vesicles. However, continued global surveillance is essential in understanding and predicting the dynamic changes in the epidemiology and biological basis of meningococcal disease and to influence the recommendations for current and future vaccines or other prevention strategies.

Translocation and Surface Expression of Lipidated Serogroup B Capsular Polysaccharide in Neisseria meningitidis

ABSTRACT The capsule of N. meningitidis serogroup B, (α2→8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal cps complex in Escherichia coli K-12 indicated that deletion of genes designated lipA and lipB caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of lip and lipB in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in lipA and lipB were generated in the serogroup B meningococcal strain NMB. The lipA and lipB genes are located on the 3′ end of the ctr operon and are most likely transcribed independently. Inactivation of lipA, lipB, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in lipA, lipB, and lipA lipB mutants. Capsular polymers purified from lipA, lipB, and lipA lipB mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus, lipA and lipB do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer.

Polymorphisms in Pilin Glycosylation Locus of Neisseria meningitidis Expressing Class II Pili

ABSTRACT We have located a locus, pgl, in Neisseria meningitidis strain NMB required for the glycosylation of class II pili. Between five and eight open reading frames (ORFs) (pglF, pglB, pglC, pglB2, orf2, orf3, orf8, and avtA) were present in the pgl clusters of different meningococcal isolates. The Class I pilus-expressing strains Neisseria gonorrhoeae MS11 and N. meningitidis MC58 each contain a pgl cluster in which orf2 andorf3 have been deleted. Strain NMB and other meningococcal isolates which express class II type IV pili contained pglclusters in which pglB had been replaced bypglB2 and an additional novel ORF, orf8, had been inserted between pglB2 and pglC. Insertional inactivation of the eight ORFs of the pglcluster of strain NMB showed that pglF, pglB2, pglC, andpglD, but not orf2, orf3, orf8, andavtA, were necessary for pilin glycosylation. Pilin glycosylation was not essential for resistance to normal human serum, as pglF and pglD mutants retained wild-type levels of serum resistance. Although pglB2 andpglC mutants were significantly sensitive to normal human serum under the experimental conditions used, subsequent examination of the encapsulation phenotypes revealed that pglB2 andpglC mutants expressed almost 50% less capsule than wild-type NMB. A mutation in orf3, which did not affect pilin glycosylation, also resulted in a 10% reduction in capsule expression and a moderately serum sensitive phenotype. On the basis of these results we suggest that pilin glycosylation may proceed via a lipid-linked oligosaccharide intermediate and that blockages in this pathway may interfere with capsular transport or assembly.

Genetic Basis for Biosynthesis of the (α1→4)-Linked N-Acetyl-d-Glucosamine 1-Phosphate Capsule of Neisseria meningitidis Serogroup X

ABSTRACT The genetic basis for biosynthesis of the (α1→4)-linked N-acetyl-d-glucosamine 1-phosphate capsule of Neisseria meningitidis serogroup X was defined. The biosynthesis gene cassette was a ∼4.2-kb region located between ctrA of the capsule transport operon and galE, which encodes the UDP-glucose-4-epimerase. This location was identical to the locations of the biosynthesis cassettes in other meningococcal serogroups. Three open reading frames unique to meningococcus serogroup X were identified. Deletion-insertion mutation and colony immunoblotting confirmed that these three genes were essential for serogroup X capsule expression, and the genes were designated xcbA, xcbB, and xcbC (serogroup Xcapsule biosynthesis). Reverse transcriptase PCR indicated that the xcbABC genes form an operon and are cotranscribed divergently from ctrA. XcbA exhibited 52% amino acid similarity to SacB, the putative capsule polymerase of meningococcus serogroup A, suggesting that it plays a role as the serogroup X capsule polymerase. An IS1016 element was found within the intergenic region separating ctrA and xcbA in multiple strains, and this element did not interfere with capsule expression.

Autoregulation of the MisR/MisS Two-Component Signal Transduction System in Neisseria meningitidis

Two-component regulatory systems are involved in processes important for bacterial pathogenesis. The proposed misR/misS (or phoP/phoQ) system is one of four two-component systems of the obligate human pathogen Neisseria meningitidis. Inactivation of this system results in loss of phosphorylation of the lipooligosaccharide inner core and causes attenuation in a mouse model of meningococcal infection. MisR and the cytoplasmic domain of MisS were purified as His6 and maltose binding protein fusion proteins, respectively. The MisS fusion was shown to be autophosphorylated in the presence of ATP, and the phosphoryl group was subsequently transferred to MisR. The phosphotransfer reaction was halted with a MisR/D52A mutation, while a MisS/H246A mutation prevented autophosphorylation. Specific interaction of phosphorylated MisR (MisR approximately P) and MisR with the misR promoter was demonstrated by gel mobility shift assays, where MisR approximately P exhibited higher affinity than did the nonphosphorylated protein. The transcriptional start site of the misRS operon was mapped, and DNase I protection assays revealed that MisR interacted with a 15-bp region upstream of the transcriptional start site that shared no similarity to binding motifs of other two-component systems. Transcriptional reporter studies suggested that MisR phosphorylation is critical for the autoinduction of the misRS operon. Limited Mg2+ concentration failed to induce expression of the misRS operon, which is the only operon now proven to be under the direct control of the MisRS two-component system. Thus, these results indicate that the meningococcal MisRS system constitutes a functional signal transduction circuit and that both components are critical in the autoregulation of their expression.