Cooperative Role for Tetraspanins in Adhesin-Mediated Attachment of Bacterial Species to Human Epithelial Cells

ABSTRACTThe tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins.Neisseria meningitidis, the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function inNeisseria meningitidisadherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence ofNeisseria meningitidis. In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells ofNeisseria meningitidisstrain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains ofStaphylococcus aureus,Neisseria lactamica,Escherichia coli, andStreptococcus pneumoniaewas also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors forNeisseria meningitidisand potentially for multiple species of bacteria.

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Meningococcal PilV Potentiates Neisseria meningitidis Type IV Pilus-Mediated Internalization into Human Endothelial and Epithelial Cells

Infection and Immunity ◽

10.1128/iai.00423-12 ◽

2012 ◽

Vol 80 (12) ◽

pp. 4154-4166 ◽

Cited By ~ 14

Author(s):

Hideyuki Takahashi ◽

Tatsuo Yanagisawa ◽

Kwang Sik Kim ◽

Shigeyuki Yokoyama ◽

Makoto Ohnishi

Keyword(s):

Mass Spectrometry ◽

Epithelial Cells ◽

Neisseria Meningitidis ◽

Host Cells ◽

Type Iv Pilus ◽

Content Type ◽

Flight Mass Spectrometry ◽

Type Iv ◽

A Minor ◽

Pilin Protein

ABSTRACTThe type IV pilus ofNeisseria meningitidisis the major factor for meningococcal adhesion to host cells. In this study, we showed that a mutant ofN. meningitidis pilV, a minor pilin protein, internalized less efficiently to human endothelial and epithelial cells than the wild-type strain. Matrix-assisted laser desorption ionization–time of flight mass spectrometry and electrospray ionization tandem mass spectrometry analyses showed that PilE, the major subunit of pili, was less glycosylated at its serine 62 residue (Ser62) in the ΔpilVmutant than in thepilV+strain, whereas phosphoglycerol at PilE Ser93 and phosphocholine at PilE Ser67 were not changed. Introduction of thepglLmutation, which results in complete loss of O-linked glycosylation from Ser62, slightly reducedN. meningitidisinternalization into human brain microvascular endothelial cells, whereas the addition of the ΔpilVmutation greatly reducedN. meningitidisinternalization. The accumulation of ezrin, which is part of the cytoskeleton ERM family, was observed withpilV+, ΔpglL, andpilE(S62A) strains but not with the ΔpilVmutant. These results suggested that whereasN. meningitidispilin originally had an adhesive activity that was less affected by minor pilin proteins, the invasive function evolved with incorporation of the PilV protein into the pili to promote theN. meningitidisinternalization into human cells.

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Prediction of Acquired Antimicrobial Resistance for Multiple Bacterial Species Using Neural Networks

mSystems ◽

10.1128/msystems.00774-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 6

Author(s):

D. Aytan-Aktug ◽

P. T. L. C. Clausen ◽

V. Bortolaia ◽

F. M. Aarestrup ◽

O. Lund

Keyword(s):

Machine Learning ◽

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Antimicrobial Resistance ◽

Bacterial Species ◽

Individual Species ◽

Learning Models ◽

Content Type ◽

Multiple Species ◽

Machine Learning Models

ABSTRACT Machine learning has proven to be a powerful method to predict antimicrobial resistance (AMR) without using prior knowledge for selected bacterial species-antimicrobial combinations. To date, only species-specific machine learning models have been developed, and to the best of our knowledge, the inclusion of information from multiple species has not been attempted. The aim of this study was to determine the feasibility of including information from multiple bacterial species to predict AMR for an individual species, since this may make it easier to train and update resistance predictions for multiple species and may lead to improved predictions. Whole-genome sequence data and susceptibility profiles from 3,528 Mycobacterium tuberculosis, 1,694 Escherichia coli, 658 Salmonella enterica, and 1,236 Staphylococcus aureus isolates were included. We developed machine learning models trained by the features of the PointFinder and ResFinder programs detected to predict binary (susceptible/resistant) AMR profiles. We tested four feature representation methods to determine the most efficient way for introducing features into the models. When training the model only on the Mycobacterium tuberculosis isolates, high prediction performances were obtained for the six AMR profiles included. By adding information on ciprofloxacin from the additional 3,588 isolates, there was no reduction in performance for the other antimicrobials but an increased performance for ciprofloxacin AMR profile prediction for Mycobacterium tuberculosis and Escherichia coli. In conclusion, the species-independent models can predict multi-AMR profiles for multiple species without losing any robustness. IMPORTANCE Machine learning is a proven method to predict AMR; however, the performance of any machine learning model depends on the quality of the input data. Therefore, we evaluated different methods of representing information about mutations as well as mobilizable genes, so that the information can serve as input for a robust model. We combined data from multiple bacterial species in order to develop species-independent machine learning models that can predict resistance profiles for multiple antimicrobials and species with high performance.

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Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence

Infection and Immunity ◽

10.1128/iai.01463-15 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1501-1513 ◽

Cited By ~ 13

Author(s):

Jakob Engman ◽

Aurel Negrea ◽

Sara Sigurlásdóttir ◽

Miriam Geörg ◽

Jens Eriksson ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Posttranslational Modifications ◽

Human Cells ◽

Bacterial Attachment ◽

Polynucleotide Phosphorylase ◽

Gene Encoding ◽

Content Type ◽

Type Iv ◽

Transcriptional Changes

Neisseria meningitidisautoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3′–5′ exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is theN. meningitidisPNPase, and we characterize its role inN. meningitidispathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in anin vivomodel ofN. meningitidisinfection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence inN. meningitidis.

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Disease-Associated Neisseria meningitidis Isolates Inhibit Wound Repair in Respiratory Epithelial Cells in a Type IV Pilus-Independent Manner

Infection and Immunity ◽

10.1128/iai.02001-14 ◽

2014 ◽

Vol 82 (12) ◽

pp. 5023-5034 ◽

Cited By ~ 2

Author(s):

Xiaoyun Ren ◽

Joanna K. MacKichan

Keyword(s):

Cell Migration ◽

Neisseria Meningitidis ◽

Meningococcal Disease ◽

Wound Repair ◽

Disease Onset ◽

Independent Manner ◽

Content Type ◽

Type Iv ◽

Respiratory Epithelial

ABSTRACTNeisseria meningitidisis the causative agent of meningococcal disease. Onset of meningococcal disease can be extremely rapid and can kill within a matter of hours. However, although a much-feared pathogen,Neisseria meningitidisis frequently found in the nasopharyngeal mucosae of healthy carriers. The bacterial factors that distinguish disease- from carriage-associated meningococci are incompletely understood. Evidence suggesting that disruptions to the nasopharynx may increase the risk of acquiring meningococcal disease led us to evaluate the ability of disease- and carriage-associated meningococcal isolates to inhibit cell migration, using anin vitroassay for wound repair. We found that disease-associated isolates in our collection inhibited wound closure, while carriage-associated isolates were more variable, with many isolates not inhibiting wound repair at all. For isolates selected for further study, we found that actin morphology, such as presence of lamellipodia, correlated with cell migration. We demonstrated that multiple meningococcal virulence factors, including the type IV pili, are dispensable for inhibition of wound repair. Inhibition of wound repair was also shown to be an active process, i.e., requiring live bacteria undergoing active protein synthesis.

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Immunogenicity and Safety of Investigational MenABCWY Vaccine and of 4CMenB and MenACWY Vaccines Administered Concomitantly or Alone: a Phase 2 Randomized Study of Adolescents and Young Adults

mSphere ◽

10.1128/msphere.00553-21 ◽

2021 ◽

Author(s):

Jiří Beran ◽

Daniel Dražan ◽

Igwebuike Enweonye ◽

Chiranjiwi Bhusal ◽

Daniela Toneatto

Keyword(s):

Young Adults ◽

Neisseria Meningitidis ◽

Bacterial Species ◽

Randomized Study ◽

Adolescents And Young Adults ◽

Phase 2 ◽

Content Type

The bacterial species Neisseria meningitidis is a major cause of meningitis, with six meningococcal groups (serogroups) causing most cases. A licensed vaccine, MenACWY (Menveo), targets four of these meningococcal serogroups, and another vaccine, 4CMenB (Bexsero), targets serogroup B.

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Discovery of a New Neisseria gonorrhoeae Type IV Pilus Assembly Factor, TfpC

mBio ◽

10.1128/mbio.02528-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Linda I. Hu ◽

Shaohui Yin ◽

Egon A. Ozer ◽

Lee Sewell ◽

Saima Rehman ◽

...

Keyword(s):

Cell Surface ◽

Neisseria Gonorrhoeae ◽

Bacterial Cell ◽

Bacterial Species ◽

Type Iv Pili ◽

Type Iv Pilus ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Type Iv

ABSTRACT Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain. IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.

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PathogenicMannheimia haemolyticaInvades Differentiated Bovine Airway Epithelial Cells

Infection and Immunity ◽

10.1128/iai.00078-19 ◽

2019 ◽

Vol 87 (6) ◽

Cited By ~ 8

Author(s):

Daniel Cozens ◽

Erin Sutherland ◽

Miquel Lauder ◽

Geraldine Taylor ◽

Catherine C. Berry ◽

...

Keyword(s):

Epithelial Cells ◽

Respiratory Tract ◽

Bacterial Species ◽

Airway Epithelial Cells ◽

Cellular Damage ◽

Economic Losses ◽

Bacterial Invasion ◽

Airway Epithelial ◽

Content Type ◽

Proinflammatory Mediators

ABSTRACTThe Gram-negative bacteriumMannheimia haemolyticais the primary bacterial species associated with bovine respiratory disease (BRD) and is responsible for significant economic losses to livestock industries worldwide. Healthy cattle are frequently colonized by commensal serotype A2 strains, but disease is usually caused by pathogenic strains of serotype A1. For reasons that are poorly understood, a transition occurs within the respiratory tract and a sudden explosive proliferation of serotype A1 bacteria leads to the onset of pneumonic disease. Very little is known about the interactions ofM. haemolyticawith airway epithelial cells of the respiratory mucosa which might explain the different abilities of serotype A1 and A2 strains to cause disease. In the present study, host-pathogen interactions in the bovine respiratory tract were mimicked using a novel differentiated bovine bronchial epithelial cell (BBEC) infection model. In this model, differentiated BBECs were inoculated with serotype A1 or A2 strains ofM. haemolyticaand the course of infection followed over a 5-day period by microscopic assessment and measurement of key proinflammatory mediators. We have demonstrated that serotype A1, but not A2,M. haemolyticainvades differentiated BBECs by transcytosis and subsequently undergoes rapid intracellular replication before spreading to adjacent cells and causing extensive cellular damage. Our findings suggest that the explosive proliferation of serotype A1M. haemolyticathat occurs within the bovine respiratory tract prior to the onset of pneumonic disease is potentially due to bacterial invasion of, and rapid proliferation within, the mucosal epithelium. The discovery of this previously unrecognized mechanism of pathogenesis is important because it will allow the serotype A1-specific virulence determinants responsible for invasion to be identified and thereby provide opportunities for the development of new strategies for combatting BRD aimed at preventing early colonization and infection of the bovine respiratory tract.

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Inactivation of NMB0419, Encoding a Sel1-Like Repeat (SLR) Protein, in Neisseria meningitidis Is Associated with Differential Expression of Genes Belonging to the Fur Regulon and Reduced Intraepithelial Replication

Infection and Immunity ◽

10.1128/iai.00574-16 ◽

2017 ◽

Vol 85 (5) ◽

Cited By ~ 1

Author(s):

Ming-Shi Li ◽

Paul R. Langford ◽

J. Simon Kroll

Keyword(s):

Epithelial Cells ◽

Neisseria Meningitidis ◽

Iron Acquisition ◽

Regulatory Function ◽

Growth Defect ◽

Wild Type ◽

Content Type ◽

Expression Of Genes ◽

New Perspective

ABSTRACT Neisseria meningitidis is a commensal microbe that colonizes the human nasopharynx but occasionally invades the bloodstream to cause life-threatening infection. N. meningitidis MC58 NMB0419 encodes a Sel1-like repeat (SLR)-containing protein, previously implicated in invasion of epithelial cells. A gene-regulatory function was revealed in Escherichia coli expressing plasmid-borne NMB0419 and showing significantly increased epithelial adherence compared to the wild type, due to increased expression of mannose-sensitive type 1 pili. While a meningococcal NMB0419 mutant did not have altered epithelial adherence, in a transcriptome-wide comparison of the wild type and an NMB0419 mutant, a large proportion of genes differentially regulated in the mutant were involved in iron acquisition and metabolism. Fifty-one percent and 38% of genes, respectively, up- and downregulated in the NMB0419 mutant had previously been identified as being induced and repressed by meningococcal Fur. An in vitro growth defect of the NMB0419 mutant under iron restriction was consistent with the downregulation of tbpAB and hmbR, while an intraepithelial replication defect was consistent with the downregulation of tonB, exbB, and exbD, based on a known phenotype of a meningococcal tonB mutant. Disruption of the N-terminal NMB0419 signal peptide, predicted to export the protein beyond the cytoplasmic membrane, resulted in loss of functional traits in N. meningitidis and E. coli. Our study indicates that the expression of NMB0419 is associated with transcriptional changes counterbalancing the regulatory function of Fur, offering a new perspective on regulatory mechanisms involved in meningococcal interaction with epithelial cells, and suggests new insights into the roles of SLR-containing genes in other bacteria.

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Disease and Carrier Isolates of Neisseria meningitidis Cause G1Cell Cycle Arrest in Human Epithelial Cells

Infection and Immunity ◽

10.1128/iai.00296-16 ◽

2016 ◽

Vol 84 (10) ◽

pp. 2758-2770 ◽

Cited By ~ 8

Author(s):

Michael von Papen ◽

Wilhelm F. Oosthuysen ◽

Jérôme Becam ◽

Heike Claus ◽

Alexandra Schubert-Unkmeir

Keyword(s):

Cell Cycle ◽

Epithelial Cells ◽

Neisseria Meningitidis ◽

Host Cell ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

Microbial Pathogens ◽

Meningococcal Infection ◽

Content Type ◽

Cyclin Dependent Kinase Inhibitor

Microbial pathogens have developed several mechanisms to modulate and interfere with host cell cycle progression. In this study, we analyzed the effect of the human pathogenNeisseria meningitidison the cell cycle of epithelial cells. Two pathogenic isolates, as well as two carrier isolates, were tested for their ability to adhere to and invade into the epithelial cell lines Detroit 562 and NP69 and to modulate the cell cycle. We found that all isolates adhered equally well to both Detroit 562 and NP69 cells, whereas the carrier isolates were significantly less invasive. Using propidium iodide staining and 5-ethynyl-2′-deoxyuridine pulse-labeling, we provide evidence that meningococcal infection arrested cells in the G1phase of the cell cycle at 24 h postinfection. In parallel, a significant decrease of cells in the S phase was observed. Interestingly, G1-phase arrest was only induced after infection with live bacteria but not with heat-killed bacteria. By Western blotting we demonstrate that bacterial infection resulted in a decreased protein level of the cell cycle regulator cyclin D1, whereas cyclin E expression levels were increased. Furthermore,N. meningitidisinfection induced an accumulation of the cyclin-dependent kinase inhibitor (CKI) p21WAF1/CIP1that was accompanied by a redistribution of this CKI to the cell nucleus, as shown by immunofluorescence analysis. Moreover, the p27CIP1CKI was redistributed and showed punctate foci in infected cells. In summary, we present data thatN. meningitidiscan interfere with the processes of host cell cycle regulation.

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New Complementation Constructs for Inducible and Constitutive Gene Expression in Neisseria gonorrhoeae and Neisseria meningitidis

Applied and Environmental Microbiology ◽

10.1128/aem.07871-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3068-3078 ◽

Cited By ~ 22

Author(s):

Meghan E. Ramsey ◽

Kathleen T. Hackett ◽

Chaitra Kotha ◽

Joseph P. Dillard

Keyword(s):

Gene Expression ◽

Neisseria Gonorrhoeae ◽

Neisseria Meningitidis ◽

Inducible Promoter ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Content Type ◽

Type Iv ◽

Low Concentrations ◽

Constitutive Gene Expression

ABSTRACTWe have created new complementation constructs for use inNeisseria gonorrhoeaeandNeisseria meningitidis. The constructs contain regions of homology with the chromosome and direct the insertion of a gene of interest into the intergenic region between the genesigaandtrpB. In order to increase the available options for gene expression inNeisseria, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-β-d-thiogalactopyranoside-induciblelacpromoter, which has been widely used inNeisseria. We also designed a construct that contains the strong, constitutive promoter from the gonococcalopaBgene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter inNeisseria. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenicNeisseriaat very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy inN. gonorrhoeaeas well as to express a translational fusion of alkaline phosphatase with TraW. TraW is a component of the gonococcal type IV secretion system, and we demonstrate that TraW localizes to the periplasm.

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