Structural Characterization of Haemophilus influenzae Enolase and Its Interaction with Human Plasminogen by In Silico and In Vitro Assays

Haemophilus influenzae is the causal agent of invasive pediatric diseases, such as meningitis, epiglottitis, pneumonia, septic arthritis, pericarditis, cellulitis, and bacteremia (serotype b). Non-typeable H. influenzae (NTHi) strains are associated with localized infections, such as otitis media, conjunctivitis, sinusitis, bronchitis, and pneumonia, and can cause invasive diseases, such as as meningitis and sepsis in immunocompromised hosts. Enolase is a multifunctional protein and can act as a receptor for plasminogen, promoting its activation to plasmin, which leads to the degradation of components of the extracellular matrix, favoring host tissue invasion. In this study, using molecular docking, three important residues involved in plasminogen interaction through the plasminogen-binding motif (251EFYNKENGMYE262) were identified in non-typeable H. influenzae enolase (NTHiENO). Interaction with the human plasminogen kringle domains is conformationally stable due to the formation of four hydrogen bonds corresponding to enoTYR253-plgGLU1 (K2), enoTYR253-plgGLY310 (K3), and enoLYS255-plgARG471/enoGLU251-plgLYS468 (K5). On the other hand, in vitro assays, such as ELISA and far-western blot, showed that NTHiENO is a plasminogen-binding protein. The inhibition of this interaction using polyclonal anti-NTHiENO antibodies was significant. With these results, we can propose that NTHiENO–plasminogen interaction could be one of the mechanisms used by H. influenzae to adhere to and invade host cells.

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Ultrastructural studies on the interaction of haemophilus influenzae with human endothelial cells in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010016073x ◽

1990 ◽

Vol 48 (3) ◽

pp. 636-637

Author(s):

D.J.P. Ferguson ◽

M. Virji ◽

H. Kayhty ◽

E.R. Moxon

Keyword(s):

Endothelial Cells ◽

Haemophilus Influenzae ◽

Intercellular Junctions ◽

Blood Stream ◽

Ultrastructural Studies ◽

Endothelial Barriers ◽

Serotype B ◽

Meningitis In Children ◽

Respiratory Epithelial

Haemophilus influenzae is a human pathogen which causes meningitis in children. Systemic H. influenzae infection is largely confined to encapsulated serotype b organisms and is a major cause of meningitis in the U.K. and elsewhere. However, the pathogenesis of the disease is still poorly understood. Studies in the infant rat model, in which intranasal challenge results in bacteraemia, have shown that H. influenzae enters submucosal tissues and disseminates to the blood stream within minutes. The rapidity of these events suggests that H. influenzae penetrates both respiratory epithelial and endothelial barriers with great efficiency. It is not known whether the bacteria penetrate via the intercellular junctions, are translocated within the cells or carried across the cellular barrier in 'trojan horse' fashion within phagocytes. In the present studies, we have challenged cultured human umbilical cord_vein endothelial cells (HUVECs) with both capsulated (b+) and capsule-deficient (b-) isogenic variants of one strain of H. influenzae in order to investigate the interaction between the bacteria and HUVEC and the effect of the capsule.

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1,3,4-Thiadiazoles Effectively Inhibit Proliferation of Toxoplasma gondii

Cells ◽

10.3390/cells10051053 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1053

Author(s):

Lidia Węglińska ◽

Adrian Bekier ◽

Katarzyna Dzitko ◽

Barbara Pacholczyk-Sienicka ◽

Łukasz Albrecht ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Direct Action ◽

Human Fibroblasts ◽

Imidazole Ring ◽

Host Cells ◽

In Vitro Assays ◽

Host Cell Invasion

Congenital and acquired toxoplasmosis caused by the food- and water-born parasite Toxoplasma gondii (T. gondii) is one of the most prevalent zoonotic infection of global importance. T. gondii is an obligate intracellular parasite with limited capacity for extracellular survival, thus a successful, efficient and robust host cell invasion process is crucial for its survival, proliferation and transmission. In this study, we screened a series of novel 1,3,4-thiadiazole-2-halophenylamines functionalized at the C5 position with the imidazole ring (1b–12b) for their effects on T. gondii host cell invasion and proliferation. To achieve this goal, these compounds were initially subjected to in vitro assays to assess their cytotoxicity on human fibroblasts and then antiparasitic efficacy. Results showed that all of them compare favorably to control drugs sulfadiazine and trimethoprim in terms of T. gondii growth inhibition (IC50) and selectivity toward the parasite, expressed as selectivity index (SI). Subsequently, the most potent of them with meta-fluoro 2b, meta-chloro 5b, meta-bromo 8b, meta-iodo 11b and para-iodo 12b substitution were tested for their efficacy in inhibition of tachyzoites invasion and subsequent proliferation by direct action on established intracellular infection. All the compounds significantly inhibited the parasite invasion and intracellular proliferation via direct action on both tachyzoites and parasitophorous vacuoles formation. The most effective was para-iodo derivative 12b that caused reduction in the percentage of infected host cells by 44% and number of tachyzoites per vacuole by 93% compared to non-treated host cells. Collectively, these studies indicate that 1,3,4-thiadiazoles 1b–12b, especially 12b with IC50 of 4.70 µg/mL and SI of 20.89, could be considered as early hit compounds for future design and synthesis of anti-Toxoplasma agents that effectively and selectively block the invasion and subsequent proliferation of T. gondii into host cells.

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A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus

Access Microbiology ◽

10.1099/acmi.ac2020.po0394 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Shauna D. Drumm ◽

Rebecca Owens ◽

Jennifer Mitchell ◽

Orla M. Keane

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Surface Proteins ◽

Host Cells ◽

Mass Spectrometry Analysis ◽

In Vitro Assays ◽

Immune Recognition ◽

Independent Manner ◽

Genes Encoding

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins. Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes. Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages. These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.

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The N-terminal half of the core protein of hepatitis C virus is sufficient for nucleocapsid formation

Journal of General Virology ◽

10.1099/vir.0.79775-0 ◽

2004 ◽

Vol 85 (4) ◽

pp. 971-981 ◽

Cited By ~ 54

Author(s):

Nathalie Majeau ◽

Valérie Gagné ◽

Annie Boivin ◽

Marilène Bolduc ◽

Josée-Anne Majeau ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Host Cells ◽

Yeast Cells ◽

Main Function ◽

C Protein ◽

Multifunctional Protein ◽

The Core

The core (C) protein of hepatitis C virus (HCV) appears to be a multifunctional protein that is involved in many viral and cellular processes. Although its effects on host cells have been extensively discussed in the literature, little is known about its main function, the assembly and packaging of the viral genome. We have studied the in vitro assembly of several deleted versions of recombinant HCV C protein expressed in E. coli. We demonstrated that the 75 N-terminal residues of the C protein were sufficient to assemble and generate nucleocapsid-like particles (NLPs) in vitro. However, homogeneous particles of regular size and shape were observed only when NLPs were produced from at least the first 79 N-terminal amino acids of the C protein. This small protein unit fused to the endoplasmic reticulum-anchoring domain also generated NLPs in yeast cells. These data suggest that the N-terminal half of the C protein is important for formation of NLPs. Similarities between the HCV C protein and C proteins of other members of the Flaviviridae are discussed.

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Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk

mBio ◽

10.1128/mbio.01765-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 55

Author(s):

Buket Baddal ◽

Alessandro Muzzi ◽

Stefano Censini ◽

Raffaele A. Calogero ◽

Giulia Torricelli ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Cross Talk ◽

Induced Defense ◽

Bronchial Epithelium ◽

Host Cells ◽

Central Metabolism ◽

Nontypeable Haemophilus Influenzae ◽

Human Respiratory Tract ◽

Content Type

ABSTRACTThe ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeableHaemophilus influenzae(NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epitheliumin vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection inHaemophilusspecies. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies.IMPORTANCESimultaneous monitoring of infection-linked transcriptome alterations in an invading pathogen and its target host cells represents a key strategy for identifying regulatory responses that drive pathogenesis. In this study, we report the progressive events of NTHi colonization in a highly differentiated model of ciliated bronchial epithelium. Genome-wide transcriptome maps of NTHi during infection provided mechanistic insights into bacterial adaptive responses to the host niche, with modulation of the central metabolism as an important signature of the evolving milieu. Our data indicate that infected epithelia respond by substantial alteration of the cytoskeletal network and cytokine repertoire, revealing a dynamic cross talk that is responsible for the onset of inflammation. This work significantly enhances our understanding of the means by which NTHi promotes infection on human mucosae and reveals novel strategies exploited by this important pathogen to cause invasive disease.

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EnP1, a Microsporidian Spore Wall Protein That Enables Spores To Adhere to and Infect Host Cells In Vitro

Eukaryotic Cell ◽

10.1128/ec.00113-07 ◽

2007 ◽

Vol 6 (8) ◽

pp. 1354-1362 ◽

Cited By ~ 63

Author(s):

Timothy R. Southern ◽

Carrie E. Jolly ◽

Melissa E. Lester ◽

J. Russell Hayman

Keyword(s):

Cell Surface ◽

Host Cell ◽

Spore Wall ◽

Site Directed Mutagenesis ◽

Host Cells ◽

Binding Motif ◽

Heparin Binding ◽

Cell Infection ◽

Host Cell Surface

ABSTRACT Microsporidia are spore-forming fungal pathogens that require the intracellular environment of host cells for propagation. We have shown that spores of the genus Encephalitozoon adhere to host cell surface glycosaminoglycans (GAGs) in vitro and that this adherence serves to modulate the infection process. In this study, a spore wall protein (EnP1; Encephalitozoon cuniculi ECU01_0820) from E. cuniculi and Encephalitozoon intestinalis is found to interact with the host cell surface. Analysis of the amino acid sequence reveals multiple heparin-binding motifs, which are known to interact with extracellular matrices. Both recombinant EnP1 protein and purified EnP1 antibody inhibit spore adherence, resulting in decreased host cell infection. Furthermore, when the N-terminal heparin-binding motif is deleted by site-directed mutagenesis, inhibition of adherence is ablated. Our transmission immunoelectron microscopy reveals that EnP1 is embedded in the microsporidial endospore and exospore and is found in high abundance in the polar sac/anchoring disk region, an area from which the everting polar tube is released. Finally, by using a host cell binding assay, EnP1 is shown to bind host cell surfaces but not to those that lack surface GAGs. Collectively, these data show that given its expression in both the endospore and the exospore, EnP1 is a microsporidian cell wall protein that may function both in a structural capacity and in modulating in vitro host cell adherence and infection.

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Characterization and Growth of Polymorphic Rickettsia felis in a Tick Cell Line

Applied and Environmental Microbiology ◽

10.1128/aem.00025-08 ◽

2008 ◽

Vol 74 (10) ◽

pp. 3151-3158 ◽

Cited By ~ 34

Author(s):

Piyanate Sunyakumthorn ◽

Apichai Bourchookarn ◽

Walairat Pornwiroon ◽

Connie David ◽

Steven A. Barker ◽

...

Keyword(s):

Host Cell ◽

Short Form ◽

Horizontal Transmission ◽

Pcr Amplification ◽

Host Cells ◽

In Vitro Assays ◽

Rickettsia Felis ◽

Transmission Potential ◽

Long Form

ABSTRACT Morphological differentiation in some arthropod-borne bacteria is correlated with increased bacterial virulence, transmission potential, and/or as a response to environmental stress. In the current study, we utilized an in vitro model to examine Rickettsia felis morphology and growth under various culture conditions and bacterial densities to identify potential factors that contribute to polymorphism in rickettsiae. We utilized microscopy (electron microscopy and immunofluorescence), genomic (PCR amplification and DNA sequencing of rickettsial genes), and proteomic (Western blotting and liquid chromatography-tandem mass spectrometry) techniques to identify and characterize morphologically distinct, long-form R. felis. Without exchange of host cell growth medium, polymorphic R. felis was detected at 12 days postinoculation when rickettsiae were seeded at a multiplicity of infection (MOI) of 5 and 50. Compared to short-form R. felis organisms, no change in membrane ultrastructure in long-form polymorphic rickettsiae was observed, and rickettsiae were up to six times the length of typical short-form rickettsiae. In vitro assays demonstrated that short-form R. felis entered into and replicated in host cells faster than long-form R. felis. However, when both short- and long-form R. felis organisms were maintained in cell-free medium for 12 days, the infectivity of short-form R. felis was decreased compared to long-form R. felis organisms, which were capable of entering host cells, suggesting that long-form R. felis is more stable outside the host cell. The relationship between rickettsial polymorphism and rickettsial survivorship should be examined further as the yet undetermined route of horizontal transmission of R. felis may utilize metabolically and morphologically distinct forms for successful transmission.

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In Vitro Models That Support Adhesion Specificity in Biofilms of Oral Bacteria

Advances in Dental Research ◽

10.1177/08959374970110011401 ◽

1997 ◽

Vol 11 (1) ◽

pp. 33-42 ◽

Cited By ~ 19

Author(s):

R.P. Ellen ◽

G. Lépine ◽

P.-M. Nghiem

Keyword(s):

Matrix Protein ◽

Protein S ◽

Specific Protein ◽

In Vitro Models ◽

Oral Bacteria ◽

Host Cells ◽

In Vitro Assays ◽

Covalent Bonds ◽

Enzyme Substrate Recognition

Adhesion to adsorbed pellicles and interspecies co-adhesion to form plaque biofilms involve selective interactions of bacterial adhesins with specific receptors. Our laboratory has devised in vitro assays for co-adhesion between Actinomyces naeslundii and Streptococcus oralis or Porphyromonas gingivalis on saliva-coated mineral and hexadecane droplet substrata. P. gingivalis structures significant for co-adhesion with A. naeslundii include surface vesicles and fimbriae. A family of arginine-specific cysteine proteinases in vesicles may be involved in adherence to bacteria, to host cells, and to matrix proteins. New research from several laboratories has found that such proteinases are processed from genes encoding polyproteins containing both proteinase and hemagglutinin domains. In addition to enzyme-substrate recognition, bacterial adhesion is often determined by specific protein-peptide and lectin-carbohydrate recognition. A. naeslundii - salivary proline-rich protein, S. gordonii - salivary a-amylase, and Treponema denticola - matrix protein recognition are examples of the former. Co-adhesion of A. naeslundii and S. oralis is an example of the latter. Lactose can selectively desorb A. naeslundii cells from mixed biofilms with S. oralis, a demonstration of the significance of specificity. Although non-specific forces are probably secondary to stereochemical fit in determining the selective range of surfaces that bacteria have evolved to recognize and bind, they probably help stabilize non-covalent bonds within aligned, complementary domains.

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Menaquinone-Specific Prenyl Reductase from the Hyperthermophilic Archaeon Archaeoglobus fulgidus

Journal of Bacteriology ◽

10.1128/jb.187.6.1937-1944.2005 ◽

2005 ◽

Vol 187 (6) ◽

pp. 1937-1944 ◽

Cited By ~ 18

Author(s):

Hisashi Hemmi ◽

Yoshihiro Takahashi ◽

Kyohei Shibuya ◽

Toru Nakayama ◽

Tokuzo Nishino

Keyword(s):

High Performance ◽

Recombinant Expression ◽

Reversed Phase ◽

Host Cells ◽

Archaeoglobus Fulgidus ◽

In Vitro Assays ◽

Hyperthermophilic Archaeon ◽

Quinone Profile ◽

New Compounds

ABSTRACT Four genes that encode the homologues of plant geranylgeranyl reductase were isolated from a hyperthermophilic archaeon Archaeoglobus fulgidus, which produces menaquinone with a fully saturated heptaprenyl side chain, menaquinone-7(14H). The recombinant expression of one of the homologues in Escherichia coli led to a distinct change in the quinone profile of the host cells, although the homologue is the most distantly related to the geranylgeranyl reductase. The new compounds found in the profile had successively longer elution times than those of ordinary quinones from E. coli, i.e., menaquinone-8 and ubiquinone-8, in high-performance liquid chromatography on a reversed-phase column. Structural analyses of the new compounds by electron impact-mass spectrometry indicated that their molecular masses progressively increase relative to the ordinary quinones at a rate of 2 U but that they still contain quinone head structures, strongly suggesting that the compounds are quinones with partially saturated prenyl side chains. In vitro assays with dithionite as the reducing agent showed that the prenyl reductase is highly specific for menaquinone-7, rather than ubiquinone-8 and prenyl diphosphates. This novel enzyme noncovalently binds flavin adenine dinucleotide, similar to geranylgeranyl reductase, but was not able to utilize NAD(P)H as the electron donor, unlike the plant homologue.

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Functional Dissection of a Conserved Motif within the Pilus Retraction Protein PilT

Journal of Bacteriology ◽

10.1128/jb.187.2.611-618.2005 ◽

2005 ◽

Vol 187 (2) ◽

pp. 611-618 ◽

Cited By ~ 30

Author(s):

Kelly G. Aukema ◽

Erin M. Kron ◽

Timothy J. Herdendorf ◽

Katrina T. Forest

Keyword(s):

Host Cells ◽

Binding Motif ◽

Functional Homology ◽

Type Iv ◽

Phage Sensitivity ◽

Surface Motility ◽

Double Substitution ◽

Α Helix

ABSTRACT PilT is a hexameric ATPase required for type IV pilus retraction in gram-negative bacteria. Retraction of type IV pili mediates intimate attachment to and signaling in host cells, surface motility, biofilm formation, natural transformation, and phage sensitivity. We investigated the in vivo and in vitro roles of each amino acid of the distinct, highly conserved C-terminal AIRNLIRE motif in PilT. Substitution of amino acids A288, I289, L292, and I293 as well as a double substitution of R290 and R294 abolished Pseudomonas aeruginosa PilT function in vivo, as measured by a loss of surface motility and phage sensitivity. When introduced into purified Aquifex aeolicus PilT, substitutions in the AIRNLIRE motif did not disrupt ATPase activity or oligomerization. In contrast, a K136Q substitution in the broadly conserved nucleotide binding motif prevented PilT function in vivo as well as in vitro. We propose that the AIRNLIRE motif forms an amphipathic α helix which transmits signals between a surface-exposed protein interaction site and the ATPase core of PilT, and we recognize a potential functional homology in other type II secretion ATPases.

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