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 Phosphorylcholine Decreases Early Inflammation and Promotes the Establishment of Stable Biofilm Communities of Nontypeable Haemophilus influenzae Strain 86-028NP in a Chinchilla Model of Otitis Media

2006 ◽  
Vol 75 (2) ◽  
pp. 958-965 ◽  
Author(s):  
Wenzhou Hong ◽  
Kevin Mason ◽  
Joseph Jurcisek ◽  
Laura Novotny ◽  
Lauren O. Bakaletz ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Fluorescent Antibody ◽  
Phase Variable ◽  
Nontypeable Haemophilus Influenzae ◽  
Bacterial Surface ◽  
Culture Cells ◽  
Antibody Staining

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a leading causative agent of otitis media. Much of the inflammation occurring during NTHi disease is initiated by lipooligosaccharides (LOS) on the bacterial surface. Phosphorylcholine (PCho) is added to some LOS forms in a phase-variable manner, and these PCho+ variants predominate in vivo. Thus, we asked whether this modification confers some advantage during infection. Virulence of an otitis media isolate (NTHi strain 86-028NP) was compared with that of an isogenic PCho transferase (licD) mutant using a chinchilla (Chinchilla lanigera) model of otitis media. Animals infected with NTHi 86-028NP licD demonstrated increased early inflammation and a delayed increase in bacterial counts compared to animals infected with NTHi 86-028NP. LOS purified from chinchilla-passed NTHi 86-028NP had increased PCho content compared to LOS purified from the inoculum. Both strains were recovered from middle ear fluids as long as 14 days postinfection. Biofilms were macroscopically visible in the middle ears of euthanized animals infected with NTHi 86-028NP 7 days and 14 days postchallenge. Conversely, less dense biofilms were observed in animals infected with NTHi 86-028NP licD 7 days postinfection, and none of the animals infected with NTHi 86-028NP licD had a visible biofilm by 14 days. Fluorescent antibody staining revealed PCho+ variants within biofilms, similar to our prior results with tissue culture cells in vitro (S. L. West-Barnette, A. Rockel, and W. E. Swords, Infect. Immun. 74:1828-1836, 2006). Animals coinfected with equal proportions of both strains had equal persistence of each strain and somewhat greater severity of disease. We thus conclude that PCho promotes NTHi infection and persistence by reducing the host inflammatory response and by promoting formation of stable biofilm communities.

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 Biofilm Growth Increases Phosphorylcholine Content and Decreases Potency of Nontypeable Haemophilus influenzae Endotoxins

2006 ◽  
Vol 74 (3) ◽  
pp. 1828-1836 ◽  
Author(s):  
Shayla West-Barnette ◽  
Andrea Rockel ◽  
W. Edward Swords
Keyword(s):  
Haemophilus Influenzae ◽  
Inflammatory Responses ◽  
Opportunistic Pathogen ◽  
Host Responses ◽  
Toll Like Receptor ◽  
Nontypeable Haemophilus Influenzae ◽  
Toll Like Receptor 4 ◽  
Biofilm Growth ◽  
Bacterial Endotoxins ◽  
Cell Layers

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is a common respiratory commensal and opportunistic pathogen. NTHI is normally contained within the airways by host innate defenses that include recognition of bacterial endotoxins by Toll-like receptor 4 (TLR4). NTHI produces lipooligosaccharide (LOS) endotoxins which lack polymeric O side chains and which may contain host glycolipids. We recently showed that NTHI biofilms contain variants with sialylated LOS glycoforms that are essential to biofilm formation. In this study, we show that NTHI forms biofilms on epithelial cell layers. Confocal analysis revealed that sialylated variants were distributed throughout the biofilm, while variants expressing phosphorylcholine (PCho) were found within the biofilm. Consistent with this observation, PCho content of LOS purified from NTHI biofilms was increased compared to LOS from planktonic cultures. Hypothesizing that the observed changes in endotoxin composition could affect bioactivity, we compared inflammatory responses to NTHI LOS purified from biofilm and planktonic cultures. Our results show that endotoxins from biofilms induced weaker host innate responses. While we observed a minimal effect of sialylation on LOS bioactivity, there was a significant decrease in bioactivity associated with PCho substitutions. We thus conclude that biofilm growth increases the proportion of PCho+ variants in an NTHI population, resulting in a net decrease in LOS bioactivity. Thus, in addition to their well-documented resistance phenotypes, our data show that biofilm communities of NTHI bacteria contain variants that evoke less potent host responses.

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 Nontypeable Haemophilus influenzae Phase-Variable Genes During Experimental Human Nasopharyngeal Colonization

2013 ◽  
Vol 208 (5) ◽  
pp. 720-727 ◽  
Author(s):  
Jessica Poole ◽  
Eric Foster ◽  
Kathryn Chaloner ◽  
Jason Hunt ◽  
Michael P. Jennings ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Phase Variable ◽  
Nontypeable Haemophilus Influenzae ◽  
Nasopharyngeal Colonization ◽  
Variable Genes

scholarly journals The role of Dam methylation in phase variation of Haemophilus influenzae genes involved in defence against phage infection

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3361-3369 ◽  
Author(s):  
Piotr Zaleski ◽  
Marek Wojciechowski ◽  
Andrzej Piekarowicz
Keyword(s):  
Haemophilus Influenzae ◽  
Phase Variation ◽  
Phage Infection ◽  
Phase Variable ◽  
Repeat Tract ◽  
Insertional Inactivation ◽  
Phage Dna ◽  
Dam Methylation ◽  
Tetranucleotide Repeats

Haemophilus influenzae uses phase variation (PV) to modulate the activity of its defence systems against phage infection. The PV of the restriction–modification (R-M) system HindI, the main defence system against phage infection and incoming chromosomal and phage DNA in H. influenzae Rd, is driven by changes of the pentanucleotide repeat tract within the coding sequence of the hsdM gene and is influenced by lack of Dam methylation. Phase-variable resistance/sensitivity to phage infection correlates with changes in lipooligosaccharide (LOS) structure and occurs by slippage of tetranucleotide repeats within the gene lic2A, coding for a step in the biosynthesis of LOS. The lack of Dam activity destabilizes the tetranuclotide (5′-CAAT) repeat tract and increases the frequency of switching from sensitivity to resistance to phage infection more than in the opposite direction. The PV of the lgtC gene does not influence resistance or sensitivity to phage infection. Insertional inactivation of lic2A, but not lgtC or lgtF, leads to resistance to phage infection and to the same structure of the LOS as observed among phase-variable phage-resistant variants. This indicates that in the H. influenzae Rd LOS only the first two sugars (Glc-Gal) extending from the third heptose are part of bacterial phage receptors.

scholarly journals Relationship between Azithromycin Susceptibility and Administration Efficacy for Nontypeable Haemophilus influenzae Respiratory Infection

2015 ◽  
Vol 59 (5) ◽  
pp. 2700-2712 ◽  
Author(s):  
Begoña Euba ◽  
Javier Moleres ◽  
Cristina Viadas ◽  
Montserrat Barberán ◽  
Lucía Caballero ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Respiratory Infection ◽  
Opportunistic Pathogen ◽  
Lavage Fluid ◽  
Cellular Level ◽  
Chronic Obstructive ◽  
Therapeutic Effects ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type ◽  
Anti Inflammatory

ABSTRACTNontypeableHaemophilus influenzae(NTHI) is an opportunistic pathogen that is an important cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). COPD is an inflammatory disease of the airways, and exacerbations are acute inflammatory events superimposed on this background of chronic inflammation. Azithromycin (AZM) is a macrolide antibiotic with antibacterial and anti-inflammatory properties and a clinically proven potential for AECOPD prevention and management. Relationships between AZM efficacy and resistance by NTHI and between bactericidal and immunomodulatory effects on NTHI respiratory infection have not been addressed. In this study, we employed two pathogenic NTHI strains with different AZM susceptibilities (NTHI 375 [AZM susceptible] and NTHI 353 [AZM resistant]) to evaluate the prophylactic and therapeutic effects of AZM on the NTHI-host interplay. At the cellular level, AZM was bactericidal toward intracellular NTHI inside alveolar and bronchial epithelia and alveolar macrophages, and it enhanced NTHI phagocytosis by the latter cell type. These effects correlated with the strain MIC of AZM and the antibiotic dose. Additionally, the effect of AZM on NTHI infection was assessed in a mouse model of pulmonary infection. AZM showed both preventive and therapeutic efficacies by lowering NTHI 375 bacterial counts in lungs and bronchoalveolar lavage fluid (BALF) and by reducing histopathological inflammatory lesions in the upper and lower airways of mice. Conversely, AZM did not reduce bacterial loads in animals infected with NTHI 353, in which case a milder anti-inflammatory effect was also observed. Together, the results of this work link the bactericidal and anti-inflammatory effects of AZM and frame the efficacy of this antibiotic against NTHI respiratory infection.

scholarly journals Phase-variable bacterial loci: how bacteria gamble to maximise fitness in changing environments

2019 ◽  
Vol 47 (4) ◽  
pp. 1131-1141 ◽  
Author(s):  
Zachary N. Phillips ◽  
Greg Tram ◽  
Kate L. Seib ◽  
John M. Atack
Keyword(s):  
Phase Variation ◽  
Dna Methyltransferases ◽  
Phase Variable ◽  
Environmental Condition ◽  
Bacterial Surface ◽  
Sense And Respond ◽  
Encode Phase ◽  
Variable Genes ◽  
Ideal Vaccine

Abstract Phase-variation of genes is defined as the rapid and reversible switching of expression — either ON-OFF switching or the expression of multiple allelic variants. Switching of expression can be achieved by a number of different mechanisms. Phase-variable genes typically encode bacterial surface structures, such as adhesins, pili, and lipooligosaccharide, and provide an extra contingency strategy in small-genome pathogens that may lack the plethora of ‘sense-and-respond’ gene regulation systems found in other organisms. Many bacterial pathogens also encode phase-variable DNA methyltransferases that control the expression of multiple genes in systems called phasevarions (phase-variable regulons). The presence of phase-variable genes allows a population of bacteria to generate a number of phenotypic variants, some of which may be better suited to either colonising certain host niches, surviving a particular environmental condition and/or evading an immune response. The presence of phase-variable genes complicates the determination of an organism's stably expressed antigenic repertoire; many phase-variable genes are highly immunogenic, and so would be ideal vaccine candidates, but unstable expression due to phase-variation may allow vaccine escape. This review will summarise our current understanding of phase-variable genes that switch expression by a variety of mechanisms, and describe their role in disease and pathobiology.

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.

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