Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion

Transposon mutagenesis has been applied to a hyper-invasive clinical isolate of Campylobacter jejuni, 01/51. A random transposon mutant library was screened in an in vitro assay of invasion and 26 mutants with a significant reduction in invasion were identified. Given that the invasion potential of C. jejuni is relatively poor compared to other enteric pathogens, the use of a hyper-invasive strain was advantageous as it greatly facilitated the identification of mutants with reduced invasion. The location of the transposon insertion in 23 of these mutants has been determined; all but three of the insertions are in genes also present in the genome-sequenced strain NCTC 11168. Eight of the mutants contain transposon insertions in one region of the genome (∼14 kb), which when compared with the genome of NCTC 11168 overlaps with one of the previously reported plasticity regions and is likely to be involved in genomic variation between strains. Further characterization of one of the mutants within this region has identified a gene that might be involved in adhesion to host cells.

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Detection and Characterization of Autoagglutination Activity by Campylobacter jejuni

Infection and Immunity ◽

10.1128/iai.68.11.6168-6175.2000 ◽

2000 ◽

Vol 68 (11) ◽

pp. 6168-6175 ◽

Cited By ~ 61

Author(s):

Naoaki Misawa ◽

Martin J. Blaser

Keyword(s):

Campylobacter Jejuni ◽

Natural Transformation ◽

Host Cells ◽

Vitro Assay ◽

Sodium Metaperiodate ◽

Bacterial Hydrophobicity ◽

Phosphate Buffered Saline ◽

Common Cell

ABSTRACT In several gram-negative bacterial pathogens, autoagglutination (AAG) activity is a marker for interaction with host cells and virulence. Campylobacter jejuni strains also show AAG, but this property varies considerably among strains. To examine the characteristics of C. jejuni AAG, we developed a quantitative in vitro assay. For strain 81-176, which shows high AAG, activity was optimal for cells grown for ≤24 h, was independent of growth temperature, and was best measured for cells suspended in phosphate-buffered saline at 25°C for 24 h. AAG activity was heat labile and was abolished by pronase or acid-glycine (pH 2.2) treatment but not by lipase, DNase, or sodium metaperiodate. Strain 4182 has low AAG activity, but extraction with water increased AAG, suggesting the loss of an inhibitor. Strain 6960 has weak AAG with no effect due to water extraction. Our study with clinical isolates suggests that C. jejuni strains may be grouped into three AAG phenotypes. A variant derived from strain 81116 that is flagellate but immotile showed the strong AAG exhibited by the parent strain, suggesting that motility per se is not necessary for the AAG activity. AAG correlated with both bacterial hydrophobicity and adherence to INT407 cells. Mutants which lack flagella (flaA,flaB, and flbA) or common cell surface antigen (peb1A) were constructed in strain 81-176 by natural transformation-mediated allelic exchange. Both AAG activity and bacterial hydrophobicity were abolished in the aflagellate mutants but not the peb1A mutant. In total, these findings indicate that C. jejuni AAG is highly associated with flagellar expression.

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Cytadherence-Deficient Mutants of Mycoplasma gallisepticum Generated by Transposon Mutagenesis

Infection and Immunity ◽

10.1128/iai.71.7.3812-3820.2003 ◽

2003 ◽

Vol 71 (7) ◽

pp. 3812-3820 ◽

Cited By ~ 33

Author(s):

Sigalit Mudahi-Orenstein ◽

Sharon Levisohn ◽

Steven J. Geary ◽

David Yogev

Keyword(s):

Fibroblast Cell ◽

Mycoplasma Gallisepticum ◽

Transposon Mutagenesis ◽

Model Systems ◽

Host Cells ◽

Original Strain ◽

Human Lung Fibroblast ◽

Transposon Insertion

ABSTRACT Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gmr HA-negative (HA−) colonies displaying a stable HA− phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA− mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA− mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.

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Campylobacter jejuni proteins Cj0952c and Cj0951c affect chemotactic behaviour towards formic acid and are important for invasion of host cells

Microbiology ◽

10.1099/mic.0.039438-0 ◽

2010 ◽

Vol 156 (10) ◽

pp. 3123-3135 ◽

Cited By ~ 41

Author(s):

A. Malik Tareen ◽

Javid Iqbal Dasti ◽

Andreas E. Zautner ◽

Uwe Groß ◽

Raimond Lugert

Keyword(s):

Formic Acid ◽

Campylobacter Jejuni ◽

Formate Dehydrogenase ◽

Bacterial Pathogen ◽

Host Cells ◽

Industrialized Countries ◽

Transposon Insertion ◽

Food Borne ◽

Strain Nctc ◽

Chemotactic Behaviour

Campylobacter jejuni, an important food-borne bacterial pathogen in industrialized countries and in the developing world, is one of the major causes of bacterial diarrhoea. To identify genes which are important for the invasion of host cells by the pathogen, we screened altogether 660 clones of a transposon-generated mutant library based on the clinical C. jejuni isolate B2. Thereby, we identified a clone with a transposon insertion in gene cj0952c. As in the well-characterized C. jejuni strain NCTC 11168, the corresponding protein together with the gene product of the adjacent gene cj0951c consists of two transmembrane domains, a HAMP domain and a putative MCP domain, which together are thought to act as a chemoreceptor, designated Tlp7. In this report we show that genes cj0952c and cj0951c (i) are important for the host cell invasion of the pathogen, (ii) are not translated as one protein in C. jejuni isolate B2, contradicting the idea of a postulated read-through mechanism, (iii) affect the motility of C. jejuni, (iv) alter the chemotactic behaviour of the pathogen towards formic acid, and (v) are not related to the utilization of formic acid by formate dehydrogenase.

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Development of Transposon Mutagenesis for Chlamydia muridarum

Journal of Bacteriology ◽

10.1128/jb.00366-19 ◽

2019 ◽

Vol 201 (23) ◽

Cited By ~ 5

Author(s):

Yibing Wang ◽

Scott D. LaBrie ◽

Steven J. Carrell ◽

Robert J. Suchland ◽

Zoe E. Dimond ◽

...

Keyword(s):

Genetic Manipulation ◽

Transposon Mutagenesis ◽

Content Type ◽

Chlamydia Muridarum ◽

Transposon Mutants ◽

Transposon Insertions ◽

Glycogen Biosynthesis

ABSTRACT Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum. The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements for Chlamydia infection in vivo. IMPORTANCE Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted Chlamydia species that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis. We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.

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In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Journal of Virology ◽

10.1128/jvi.77.6.3669-3679.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3669-3679 ◽

Cited By ~ 85

Author(s):

Caterina Trozzi ◽

Linda Bartholomew ◽

Alessandra Ceccacci ◽

Gabriella Biasiol ◽

Laura Pacini ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Serine Protease ◽

In Vitro Selection ◽

Three Dimensional ◽

Host Cells ◽

Dimensional Structure ◽

In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

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Characterization of an ineffective actinorhizal microsymbiont, Frankia sp. EuI1 (Actinomycetales)

Canadian Journal of Microbiology ◽

10.1139/m80-180 ◽

1980 ◽

Vol 26 (9) ◽

pp. 1072-1089 ◽

Cited By ~ 75

Author(s):

Dwight Baker ◽

William Newcomb ◽

John G. Torrey

Keyword(s):

Host Range ◽

Nitrogenase Activity ◽

Root Nodules ◽

Host Cells ◽

Vesicle Formation ◽

Nitrogen Fixing ◽

Elaeagnus Umbellata

The actinomycete, Frankia sp. EuI1, isolated from root nodules of Elaeagnus umbellata is an infective endophyte but which lacks the ability to form an effective nitrogen-fixing symbiosis with its host. This ineffective organism can be distinguished easily from other frankiae, in vitro, on the basis of size, morphology, and the elaboration of a diffusible pigment. Cross-inoculation studies indicated that the host range of this symbiont is narrow and probably restricted to the Elaeagnaceae. In all cases of nodulation the symbiosis never developed nitrogenase activity and the microsymbiont never produced endophytic vesicles within the infected host cells. Sporangia were produced in vivo and in vitro so the morphogenetic block is apparently restricted to vesicle formation.

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Bifidobacterium longum subsp. infantis ATCC 15697 and Goat Milk Oligosaccharides Show Synergism In Vitro as Anti-Infectives against Campylobacter jejuni

Foods ◽

10.3390/foods9030348 ◽

2020 ◽

Vol 9 (3) ◽

pp. 348 ◽

Cited By ~ 2

Author(s):

Erinn M. Quinn ◽

Helen Slattery ◽

Dan Walsh ◽

Lokesh Joshi ◽

Rita M. Hickey

Keyword(s):

Campylobacter Jejuni ◽

Bifidobacterium Longum ◽

Goat Milk ◽

Host Cells ◽

Intestinal Cells ◽

Milk Oligosaccharides ◽

Bifidobacterium Strain ◽

Ht 29 ◽

Human Intestinal Cells

Bifidobacteria are known to inhibit, compete with and displace the adhesion of pathogens to human intestinal cells. Previously, we demonstrated that goat milk oligosaccharides (GMO) increased the attachment of Bifidobacterium longum subsp. infantis ATCC 15697 to intestinal cells in vitro. In this study, we aimed to exploit this effect as a mechanism for inhibiting pathogen association with intestinal cells. We examined the synergistic effect of GMO-treated B. infantis on preventing the attachment of a highly invasive strain of Campylobacter jejuni to intestinal HT-29 cells. The combination decreased the adherence of C. jejuni to the HT-29 cells by an average of 42% compared to the control (non-GMO treated B. infantis). Increasing the incubation time of the GMO with the Bifidobacterium strain resulted in the strain metabolizing the GMO, correlating with a subsequent 104% increase in growth over a 24 h period when compared to the control. Metabolite analysis in the 24 h period also revealed increased production of acetate, lactate, formate and ethanol by GMO-treated B. infantis. Statistically significant changes in the GMO profile were also demonstrated over the 24 h period, indicating that the strain was digesting certain structures within the pool such as lactose, lacto-N-neotetraose, lacto-N-neohexaose 3′-sialyllactose, 6′-sialyllactose, sialyllacto-N-neotetraose c and disialyllactose. It may be that early exposure to GMO modulates the adhesion of B. infantis while carbohydrate utilisation becomes more important after the bacteria have transiently colonised the host cells in adequate numbers. This study builds a strong case for the use of synbiotics that incorporate oligosaccharides sourced from goat′s milk and probiotic bifidobacteria in functional foods, particularly considering the growing popularity of formulas based on goat milk.

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Differential Requirements for VirB1 and VirB2 during Brucella abortus Infection

Infection and Immunity ◽

10.1128/iai.72.9.5143-5149.2004 ◽

2004 ◽

Vol 72 (9) ◽

pp. 5143-5149 ◽

Cited By ~ 66

Author(s):

Andreas B. den Hartigh ◽

Yao-Hui Sun ◽

David Sondervan ◽

Niki Heuvelmans ◽

Marjolein O. Reinders ◽

...

Keyword(s):

Mouse Model ◽

Brucella Abortus ◽

Persistent Infection ◽

Host Cells ◽

Intracellular Replication ◽

Bacterial Surface ◽

Type Iv

ABSTRACT The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.

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Characterization of protease activity of Nsp3 from SARS-CoV-2 and its in vitro inhibition by nanobodies

10.1101/2020.12.09.417741 ◽

2020 ◽

Author(s):

Lee A. Armstrong ◽

Sven M. Lange ◽

Virginia de Cesare ◽

Stephen P. Matthews ◽

Raja Sekar Nirujogi ◽

...

Keyword(s):

Catalytic Domain ◽

Transmembrane Protein ◽

Cross Reactivity ◽

Host Cells ◽

Alternative Methods ◽

Structural Proteins ◽

Active Protease ◽

Micromolar Range

AbstractOf the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

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Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

10.1101/2021.02.02.428995 ◽

2021 ◽

Author(s):

Tal Noy-Porat ◽

Adva Mechaly ◽

Yinon Levy ◽

Efi Makdasi ◽

Ron Alcalay ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Phage Display Library ◽

Host Cells ◽

Terminal Domain ◽

Isolation And Characterization ◽

Recent Emergence ◽

Therapeutic Monoclonal Antibodies

AbstractSince the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

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