scholarly journals Underlying Glycans Determine the Ability of Sialylated Lipooligosaccharide To Protect Nontypeable Haemophilus influenzae from Serum IgM and Complement

2019 ◽  
Vol 87 (11) ◽  
Author(s):  
Mary Darby Jackson ◽  
Sandy M. Wong ◽  
Brian J. Akerley
Keyword(s):  
Sialic Acid ◽  
Haemophilus Influenzae ◽  
Phase Variation ◽  
Population Diversity ◽  
Classical Pathway ◽  
Immune Recognition ◽  
Phase Variable ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type ◽  
Complex Glycans

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) efficiently colonizes the human nasopharynx asymptomatically but also causes respiratory mucosal infections, including otitis media, sinusitis, and bronchitis. The lipooligosaccharide (LOS) on the cell surface of NTHi displays complex glycans that mimic host structures, allowing it to evade immune recognition. However, LOS glycans are also targets of host adaptive and innate responses. To aid in evasion of these responses, LOS structures exhibit interstrain heterogeneity and are also subject to phase variation, the random on/off switching of gene expression, generating intrastrain population diversity. Specific LOS modifications, including terminal sialylation of the LOS, which exploits host-derived sialic acid (Neu5Ac), can also block recognition of NTHi by bactericidal IgM and complement by mechanisms that are not fully understood. We investigated the LOS sialic acid-mediated resistance of NTHi to antibody-directed killing by serum complement. We identified specific LOS structures extending from heptose III that are targets for binding by naturally occurring bactericidal IgM in serum and are protected by sialylation of the LOS. Phase-variable galactosyltransferases encoded by lic2A and lgtC each add a galactose epitope bound by IgM that results in antibody-dependent killing via the classical pathway of complement. NTHi’s survival can be influenced by the expression of phase-variable structures on the LOS that may also depend on environmental conditions, such as the availability of free sialic acid. Identification of surface structures on NTHi representing potential targets for antibody-based therapies as alternatives to antibiotic treatment would thus be valuable for this medically important pathogen.

scholarly journals Closed Complete Genome Sequences of Two Nontypeable Haemophilus influenzae Strains Containing Novel modA Alleles from the Sputum of Patients with Chronic Obstructive Pulmonary Disease

2018 ◽  
Vol 7 (2) ◽  
Author(s):  
John M. Atack ◽  
Timothy F. Murphy ◽  
Lauren O. Bakaletz ◽  
Kate L. Seib ◽  
Michael P. Jennings
Keyword(s):  
Chronic Obstructive Pulmonary Disease ◽  
Haemophilus Influenzae ◽  
Pulmonary Disease ◽  
Complete Genome ◽  
Chronic Obstructive ◽  
Phase Variable ◽  
Nontypeable Haemophilus Influenzae ◽  
Genome Sequences ◽  
Obstructive Pulmonary Disease ◽  
Content Type

Nontypeable Haemophilus influenzae (NTHi) is an important bacterial pathogen that causes otitis media and exacerbations of chronic obstructive pulmonary disease (COPD). Here, we report the complete genome sequences of NTHi strains 10P129H1 and 84P36H1, isolated from COPD patients, which contain the phase-variable epigenetic regulators ModA15 and ModA18, respectively.

scholarly journals High-Depth RNA-Seq Data Sets for Studying Gene Expression Changes Mediated by Phase-Variable DNA Methyltransferases in Nontypeable Haemophilus influenzae

2019 ◽  
Vol 8 (2) ◽  
Author(s):  
John M. Atack ◽  
Lauren O. Bakaletz ◽  
Michael P. Jennings
Keyword(s):  
Haemophilus Influenzae ◽  
Bacterial Pathogen ◽  
Dna Methyltransferases ◽  
Multiple Infections ◽  
Data Sets ◽  
Phase Variable ◽  
Rna Seq ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type ◽  
High Depth

Nontypeable Haemophilus influenzae (NTHi) is a major bacterial pathogen that causes multiple infections. We report high-depth-coverage RNA-Seq data from three NTHi strains, each of which encodes a different phase-variable methyltransferase.

scholarly journals Comparative Analyses of the Lipooligosaccharides from Nontypeable Haemophilus influenzae and Haemophilus haemolyticus Show Differences in Sialic Acid and Phosphorylcholine Modifications

2016 ◽  
Vol 84 (3) ◽  
pp. 765-774 ◽  
Author(s):  
Deborah M. B. Post ◽  
Margaret R. Ketterer ◽  
Jeremy E. Coffin ◽  
Lorri M. Reinders ◽  
Robert S. Munson ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Haemophilus Influenzae ◽  
Mass Spectrometric ◽  
Lower Airway ◽  
Titration Calorimetry ◽  
Acid Uptake ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type ◽  
Comparative Analyses ◽  
Tract Infections

Haemophilus haemolyticusand nontypeableHaemophilus influenzae(NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, whileH. haemolyticusrarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent thanH. haemolyticusLOS did. Genomic analyses of 10 strains demonstrated thatH. haemolyticuslacked the sialyltransferase geneslic3Aandlic3B(9/10) andsiaA(10/10), but all strains contained the sialic acid uptake genessiaPandsiaT(10/10). However, isothermal titration calorimetry analyses of SiaP from twoH. haemolyticusstrains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS fromH. haemolyticuscontained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures fromH. haemolyticusand NTHi may explain some of the differences in their propensities to cause disease.

Mecanismos moleculares de adaptación del patógeno respiratorio Haemophilus influenzae y desarrollo de nuevos antimicrobianos

2020 ◽  
Author(s):  
◽  
Ariadna Fernández Calvet
Keyword(s):  
Haemophilus Influenzae ◽  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Phase Variation ◽  
Opportunistic Pathogen ◽  
Phase Variable ◽  
Nontypeable Haemophilus Influenzae ◽  
Molecular Systems ◽  
Bacterial Surface ◽  
Human Airways

This PhD Thesis work tackled three aspects of the interaction between the colonizing opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) and the human airways, by considering the concepts of phase-variable regulation of pathoadaptive traits (Chapter 1), the importance of molecular systems involved in maintaining the bacterial surface integrity (Chapter 2), and the therapeutic potential of xenohormetic molecules (Chapter 3). Altogether, this work contributes expanding our understanding on molecular mechanisms of NTHi pathoadaptation regulated by phase variation, provides evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules, and highlights the therapeutic potential of xenohormetic molecules against NTHi infection.

scholarly journals Phase variable glycosylation in non-typeable Haemophilus influenzae

2019 ◽  
Author(s):  
Danila Elango ◽  
Benjamin L. Schulz
Keyword(s):  
Haemophilus Influenzae ◽  
Ex Vivo ◽  
Phase Variation ◽  
Population Diversity ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Phase Variable ◽  
Repeat Elements ◽  
C Terminus ◽  
Tract Infections

AbstractNon-typeable Haemophilus influenzae (NTHi) is a leading cause of respiratory tract infections worldwide and continues to be a global health burden. Adhesion and colonisation of host cells are crucial steps in bacterial pathogenesis, and in many strains of NTHi interaction with the host is mediated by the high molecular weight adhesins HMW1A and HMW2A. These adhesins are N-glycoproteins which are modified by cytoplasmic glycosyltransferases HMW1C and HMW2C. Phase variation in the number of short sequence repeats in the promoters of hmw1A and hmw2A directly affects their expression. Here, we report the presence of similar variable repeat elements in the promoters of hmw1C and hmw2C in diverse NTHi isolates. In an ex vivo assay, we systematically altered substrate and glycosyltransferase expression and showed that both of these factors affected the site-specific efficiency of glycosylation on HMW-A. Glycosylation occupancy was incomplete at many sites, variable between sites, and generally lower close to the C-terminus of HMW-A. We investigated the causes of this variability. As HMW-C glycosylates HMW-A in the cytoplasm, we tested how secretion affected glycosylation on HMW-A and showed that retaining HMW-A in the cytoplasm indeed increased glycosylation occupancy across the full length of the protein. Site-directed mutagenesis showed that HMW-C had no inherent preference for glycosylating asparagines in NxS or NxT sequons. This work provides key insights into factors contributing to the heterogenous modifications of NTHi HMW-A adhesins, expands knowledge of NTHi population diversity and pathogenic capability, and is relevant to vaccine design for NTHi and related pathogens.

scholarly journals Shielding of a Lipooligosaccharide IgM Epitope Allows Evasion of Neutrophil-Mediated Killing of an Invasive Strain of Nontypeable Haemophilus influenzae

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Jeroen D. Langereis ◽  
Jeffrey N. Weiser
Keyword(s):  
Haemophilus Influenzae ◽  
Immune Cells ◽  
Inner Core ◽  
Inflammatory Diseases ◽  
White Blood Cells ◽  
Human Neutrophils ◽  
Chronic Obstructive ◽  
Phase Variable ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type

ABSTRACTNontypeableHaemophilus influenzaeis a frequent cause of noninvasive mucosal inflammatory diseases but may also cause invasive diseases, such as sepsis and meningitis, especially in children and the elderly. Infection by nontypeableHaemophilus influenzaeis characterized by recruitment of neutrophilic granulocytes. Despite the presence of a large number of neutrophils, infections with nontypeableHaemophilus influenzaeare often not cleared effectively by the antimicrobial activity of these immune cells. Herein, we examined how nontypeableHaemophilus influenzaeevades neutrophil-mediated killing. Transposon sequencing (Tn-seq) was used on an isolate resistant to neutrophil-mediated killing to identify genes required for its survival in the presence of human neutrophils and serum, which provided a source of complement and antibodies. Results show that nontypeableHaemophilus influenzaeprevents complement-dependent neutrophil-mediated killing by expression of surface galactose-containing oligosaccharide structures. These outer-core structures block recognition of an inner-core lipooligosaccharide epitope containing glucose attached to heptose HepIII-β1,2-Glc by replacement with galactose attached to HepIII or through shielding HepIII-β1,2-Glc by phase-variable attachment of oligosaccharide chain extensions. When the HepIII-β1,2-Glc-containing epitope is expressed and exposed, nontypeableHaemophilus influenzaeis opsonized by naturally acquired IgM generally present in human serum and subsequently phagocytosed and killed by human neutrophils. Clinical nontypeableHaemophilus influenzaeisolates containing galactose attached to HepIII that are not recognized by this IgM are more often found to cause invasive infections.IMPORTANCENeutrophils are white blood cells that specialize in killing pathogens and are recruited to sites of inflammation. However, despite the presence of large numbers of neutrophils in the middle ear cavity and lungs of patients with otitis media or chronic obstructive pulmonary disease, respectively, the bacterium nontypeableHaemophilus influenzaeis often not effectively cleared from these locations by these immune cells. In order to understand how nontypeableHaemophilus influenzaeis able to cause inflammatory diseases in the presence of neutrophils, we determined the mechanism that underlies resistance to neutrophil-mediated killing. We have shown that nontypeableHaemophilus influenzaeprevents binding of antibodies of the IgM subtype through changes in their surface lipooligosaccharide structure, thereby preventing complement activation and clearance by human neutrophils.

scholarly journals Sialic acid transport and catabolism are cooperatively regulated by SiaR and CRP in nontypeable Haemophilus influenzae

2010 ◽  
Vol 10 (1) ◽  
pp. 240 ◽  
Author(s):  
Jason W Johnston ◽  
Haider Shamsulddin ◽  
Anne-Frances Miller ◽  
Michael A Apicella
Keyword(s):  
Sialic Acid ◽  
Haemophilus Influenzae ◽  
Acid Transport ◽  
Nontypeable Haemophilus Influenzae

scholarly journals Elucidation of the Monoclonal Antibody 5G8-Reactive, Virulence-Associated Lipopolysaccharide Epitope of Haemophilus influenzae and Its Role in Bacterial Resistance to Complement-Mediated Killing

2005 ◽  
Vol 73 (4) ◽  
pp. 2213-2221 ◽  
Author(s):  
Ruth Griffin ◽  
Andrew D. Cox ◽  
Katherine Makepeace ◽  
James C. Richards ◽  
E. Richard Moxon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Haemophilus Influenzae ◽  
Bacterial Resistance ◽  
Inner Core ◽  
Phase Variation ◽  
Phase Variable ◽  
Type B ◽  
Variable Expression ◽  
Virulent Type ◽  
Unknown Structure

ABSTRACT The phase-variable locus lex2 is required for expression of a Haemophilus influenzae lipopolysaccharide (LPS) epitope of previously unknown structure. This epitope, which is reactive with monoclonal antibody (MAb) 5G8, has been associated with virulence of type b strains. When strain RM118 (from the same source as strain Rd), in which the lex2 locus and MAb 5G8 reactivity are absent, was transformed with lex2 DNA, transformants that were reactive with MAb 5G8 were obtained. Surprisingly, the 5G8 reactivity of these transformants was phase variable, although the lex2 locus lacked tetrameric repeats and was constitutively expressed. This phase variation was shown to be the result of phase-variable expression of phosphorylcholine (PCho) such that MAb 5G8 reacted only in the absence of PCho. Structural analysis showed that, compared to RM118, the lex2 transformant had acquired a tetrasaccharide, Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β1,4, linked to the proximal heptose (HepI). A terminal GalNAc was detected in a minority of glycoforms. LPS derived from a mutant of RM7004, a virulent type b strain which naturally expresses lex2 and has LPS containing the same tetrasaccharide linked to HepI as the sole oligosaccharide extension from the inner core, confirmed that GalNAc is not a part of the MAb 5G8-reactive epitope. Thus, MAb 5G8 specifically binds to the structure Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β attached via a 1,4 linkage to HepI of H. influenzae LPS, and we show that the ability to synthesize this novel tetrasaccharide was associated with enhanced bacterial resistance to complement-mediated killing.

scholarly journals Analysis of Invasive NontypeableHaemophilus influenzaeIsolates Reveals Selection for the Expression State of Particular Phase-Variable Lipooligosaccharide Biosynthetic Genes

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Zachary N. Phillips ◽  
Charles Brizuela ◽  
Amy V. Jennison ◽  
Megan Staples ◽  
Keith Grimwood ◽  
...  
Keyword(s):  
Gene Expression ◽  
Phase Variation ◽  
Phase Variable ◽  
Chronic Infections ◽  
Biosynthetic Genes ◽  
Content Type ◽  
Invasive Infections ◽  
Acetyl Group ◽  
Overt Disease ◽  
Expression Status

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a major human pathogen, responsible for several acute and chronic infections of the respiratory tract. The incidence of invasive infections caused by NTHi is increasing worldwide. NTHi is able to colonize the nasopharynx asymptomatically, and the exact change(s) responsible for transition from benign carriage to overt disease is not understood. We have previously reported that phase variation (the rapid and reversible ON-OFF switching of gene expression) of particular lipooligosaccharide (LOS) glycosyltransferases occurs during transition from colonizing the nasopharynx to invading the middle ear. Variation in the structure of the LOS is dependent on the ON/OFF expression status of each of the glycosyltransferases responsible for LOS biosynthesis. In this study, we surveyed a collection of invasive NTHi isolates for ON/OFF expression status of seven phase-variable LOS glycosyltransferases. We report that the expression state of the LOS biosynthetic genesoafAON andlic2AOFF shows a correlation with invasive NTHi isolates. We hypothesize that these gene expression changes contribute to the invasive potential of NTHi. OafA expression, which is responsible for the addition of anO-acetyl group onto the LOS, has been shown to impart a phenotype of increased serum resistance and may serve as a marker for invasive NTHi.

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