Roles of Capsule and Lipopolysaccharide O Antigen in Interactions of Human Monocyte-Derived Dendritic Cells and Klebsiella pneumoniae

ABSTRACT In humans, Klebsiella pneumoniae is a saprophytic bacterium of the nasopharyngeal and intestinal mucosae that is also frequently responsible for severe nosocomial infections. Two major factors of virulence, capsular polysaccharide (CPS) and lipopolysaccharide (LPS) O antigen, are involved in mucosal colonization and the development of infections. These bacterial surface structures are likely to play major roles in interactions with the mucosal immune system, which are orchestrated by a network of surveillance based on dendritic cells (DCs). To determine the roles of K. pneumoniae CPS and LPS in the DC response, we investigated the response of immature human monocyte-derived DCs to bacterial challenge with a wild-type strain and its isogenic mutants deficient in CPS or LPS O-antigen production. As observed by flow cytometry and confocal laser microscopy, the rate of phagocytosis was inversely proportional to the amount of CPS on the bacterial cell surface, with LPS playing little or no role. The K. pneumoniae wild-type strain induced DC maturation with upregulation of CD83, CD86, and TLR4 and downregulation of CD14 and DC-SIGN. With CPS mutants, we observed a greater decrease in DC-SIGN, suggesting a superior maturation of DCs. In addition, incubation of DCs with CPS mutants, and to a lesser extent with LPS mutants, resulted in significantly higher Th1 cytokine production. Combined, our findings suggest that K. pneumoniae CPS, by hampering bacterial binding and internalization, induces a defective immunological host response, including maturation of DCs and pro-Th1 cytokine production, whereas the LPS O antigen seems to be involved essentially in DC activation.

Download Full-text

Comparison of the Host Responses to Wild-Type and cpsB Mutant Klebsiella pneumoniae Infections

Infection and Immunity ◽

10.1128/iai.00244-06 ◽

2006 ◽

Vol 74 (9) ◽

pp. 5402-5407 ◽

Cited By ~ 45

Author(s):

Matthew S. Lawlor ◽

Scott A. Handley ◽

Virginia L. Miller

Keyword(s):

Klebsiella Pneumoniae ◽

Mouse Model ◽

Type Strain ◽

Wild Type Strain ◽

Capsular Polysaccharide ◽

Cytokine Production ◽

Host Responses ◽

Cellular Infiltration ◽

Wild Type ◽

Highly Virulent

ABSTRACT Previously, we established an intranasal mouse model of Klebsiella pneumoniae infection and validated its utility using a highly virulent wild-type strain and an avirulent capsular polysaccharide mutant. In the present study we compare the host responses to both infections by examining cytokine production, cellular infiltration, pulmonary histology, and intranasal immunization.

Download Full-text

The UDP N-Acetylgalactosamine 4-Epimerase Gene Is Essential for Mesophilic Aeromonas hydrophila Serotype O34 Virulence

Infection and Immunity ◽

10.1128/iai.74.1.537-548.2006 ◽

2006 ◽

Vol 74 (1) ◽

pp. 537-548 ◽

Cited By ~ 23

Author(s):

Rocío Canals ◽

Natalia Jiménez ◽

Silvia Vilches ◽

Miguel Regué ◽

Susana Merino ◽

...

Keyword(s):

Aeromonas Hydrophila ◽

Type Strain ◽

Wild Type Strain ◽

Sugar Residue ◽

Serum Resistance ◽

Single Mutation ◽

Wild Type ◽

O Antigen ◽

Wild Type Phenotype

ABSTRACT Mesophilic Aeromonas hydrophila strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP N-acetylgalactosamine 4-epimerase (gne) confers the O− phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The gne gene is present in all the mesophilic Aeromonas strains tested. No changes were observed for the LPS core in a gne mutant from A. hydrophila strain AH-3 (serotype O34). O34 antigen LPS contains N-acetylgalactosamine, while no such sugar residue forms part of the LPS core from A. hydrophila AH-3. Some of the pathogenic features of A. hydrophila AH-3 gne mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the gne mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic Aeromonas strains, possess two kinds of flagella, and the absence of O34 antigen molecules by gne mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type gne gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).

Download Full-text

Role of Lung Epithelial Cells in Defense against Klebsiella pneumoniae Pneumonia

Infection and Immunity ◽

10.1128/iai.70.3.1075-1080.2002 ◽

2002 ◽

Vol 70 (3) ◽

pp. 1075-1080 ◽

Cited By ~ 41

Author(s):

Guadalupe Cortés ◽

Dolores Álvarez ◽

Carles Saus ◽

Sebastián Albertí

Keyword(s):

Epithelial Cells ◽

Klebsiella Pneumoniae ◽

Type Strain ◽

Pathogenic Bacteria ◽

Wild Type Strain ◽

Capsular Polysaccharide ◽

Defense Mechanism ◽

Lung Epithelial Cells ◽

Wild Type ◽

Lung Epithelial

ABSTRACT The airway epithelium represents a primary site for the entry of pathogenic bacteria into the lungs. It has been suggested for many respiratory pathogens, including Klebsiella pneumoniae, that adhesion and invasion of the lung epithelial cells is an early stage of the pneumonia process. We observed that poorly encapsulated K. pneumoniae clinical isolates and an isogenic unencapsulated mutant invaded lung epithelial cells more efficiently than highly encapsulated strains independent of the K type. By contrast, the unencapsulated mutant was completely avirulent in a mouse model of pneumonia, unlike the wild-type strain, which produced pneumonia and systemic infection. Furthermore, the unencapsulated mutant bound more epithelially produced complement component C3 than the wild-type strain. Our results show that lung epithelial cells play a key role as a host defense mechanism against K. pneumoniae pneumonia, using two different strategies: (i) ingestion and control of the microorganisms and (ii) opsonization of the microorganisms. Capsular polysaccharide avoids both mechanisms and enhances the virulence of K. pneumoniae.

Download Full-text

Role of β-Lactamases and Porins in Resistance to Ertapenem and Other β-Lactams in Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.8.3203-3206.2004 ◽

2004 ◽

Vol 48 (8) ◽

pp. 3203-3206 ◽

Cited By ~ 127

Author(s):

George A. Jacoby ◽

Debra M. Mills ◽

Nancy Chow

Keyword(s):

Klebsiella Pneumoniae ◽

Outer Membrane ◽

Type Strain ◽

Wild Type Strain ◽

Outer Membrane Proteins ◽

Wild Type ◽

Resistant Mutants ◽

High Level ◽

Level Resistance

ABSTRACT High-level resistance to ertapenem was produced by β-lactamases of groups 1, 2f, and 3 in a strain of Klebsiella pneumoniae deficient in Omp35 and Omp36. From a wild-type strain producing ACT-1 β-lactamase, ertapenem-resistant mutants for which the ertapenem MICs were up to 128 μg/ml and expression of outer membrane proteins was diminished could be selected.

Download Full-text

Shigella flexneri IpaH7.8 Facilitates Escape of Virulent Bacteria from the Endocytic Vacuoles of Mouse and Human Macrophages

Infection and Immunity ◽

10.1128/iai.68.6.3608-3619.2000 ◽

2000 ◽

Vol 68 (6) ◽

pp. 3608-3619 ◽

Cited By ~ 57

Author(s):

Carmen M. Fernandez-Prada ◽

David L. Hoover ◽

Ben D. Tall ◽

Antoinette B. Hartman ◽

June Kopelowitz ◽

...

Keyword(s):

Type Strain ◽

Deletion Mutant ◽

Wild Type Strain ◽

Shigella Flexneri ◽

Human Monocyte ◽

Avirulent Strain ◽

Human Monocytes ◽

Wild Type ◽

Distribution Profile ◽

J774 Cells

ABSTRACT The behavior of Shigella flexneri ipaH mutants was studied in human monocyte-derived macrophages (HMDM), in 1-day-old human monocytes, and in J774 mouse macrophage cell line. In HMDM, strain pWR700, an ipaH 7.8 deletion mutant ofS. flexneri 2a strain 2457T, behaved like the wild-type strain 2457T. This strain caused rapid host cell death by oncosis, and few bacterial CFU were recovered after incubation in the presence of gentamicin as previously described for 2457T-infected HMDM. However, analysis of bacterial compartmentalization within endocytic vacuoles with gentamicin and chloroquine indicated that more pWR700 than 2457T was present within the endocytic vacuoles of HMDM, suggesting thatipaH 7.8 deletion mutant transited more slowly from the vacuoles to the cytoplasm. In contrast to findings with HMDM, CFU recovered from pWR700-infected mouse J774 cells were 2 to 3 logs higher than CFU from 2457T-infected J774 cells. These values exceeded CFU recovered after infection of J774 cells with plasmid-cured avirulent strain M4243A1. Incubation with gentamicin and chloroquine clearly showed that pWR700 within J774 cells was mostly present within the endocytic vacuoles. This distribution pattern was similar to that seen with M4243A1 and contrasted with the pattern seen with 2457T. Complementation of pWR700 with a recombinant clone expressingipaH 7.8 restored the intracellular distribution of bacteria to that seen with the wild-type strain. Strains with deletions in ipaH 4.5 oripaH 9.8, however, behaved like 2457T in both HMDM and J774 cells. The distribution profile of pWR700 in 1-day-old monocytes was similar to that seen in J774 cells. Like infected J774 cells, 1-day-old human monocytes demonstrated apoptosis upon infection with virulent Shigella. These results suggest that a role of the ipaH 7.8 gene product is to facilitate the escape of the virulent bacteria from the phagocytic vacuole of monocytes and macrophages.

Download Full-text

Cell-to-Cell Signaling in Xylella fastidiosa Suppresses Movement and Xylem Vessel Colonization in Grape

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-10-1309 ◽

2008 ◽

Vol 21 (10) ◽

pp. 1309-1315 ◽

Cited By ~ 71

Author(s):

Subhadeep Chatterjee ◽

Karyn L. Newman ◽

Steven E. Lindow

Keyword(s):

Cell Signaling ◽

Type Strain ◽

Laser Scanning ◽

Wild Type Strain ◽

Xylella Fastidiosa ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Deficient Mutant ◽

Wild Type ◽

Wide Range

Cell-to-cell signaling mediated by a fatty acid diffusible signaling factor (DSF) is central to the regulation of the virulence of Xylella fastidiosa. DSF production by X. fastidiosa is dependent on rpfF and, although required for insect colonization, appears to reduce its virulence to grape. To understand what aspects of colonization of grape are controlled by DSF in X. fastidiosa and, thus, those factors that contribute to virulence, we assessed the colonization of grape by a green fluorescent protein–marked rpfF-deficient mutant. The rpfF-deficient mutant was detected at a greater distance from the point of inoculation than the wild-type strain at a given sampling time, and also attained a population size that was up to 100-fold larger than that of the wild-type strain at a given distance from the point of inoculation. Confocal laser-scanning microscopy revealed that approximately 10-fold more vessels in petioles of symptomatic leaves harbored at least some cells of either the wild type or rpfF mutant when compared with asymptomatic leaves and, thus, that disease symptoms were associated with the extent of vessel colonization. Importantly, the rpfF mutant colonized approximately threefold more vessels than the wild-type strain. Although a wide range of colony sizes were observed in vessels colonized by both the wild type and rpfF mutant, the proportion of colonized vessels harboring large numbers of cells was significantly higher in plants inoculated with the rpfF mutant than with the wild-type strain. These studies indicated that the hypervirulence phenotype of the rpfF mutant is due to both a more extensive spread of the pathogen to xylem vessels and unrestrained multiplication within vessels leading to blockage. These results suggest that movement and multiplication of X. fastidiosa in plants are linked, perhaps because cell wall degradation products are a major source of nutrients. Thus, DSF-mediated cell-to-cell signaling, which restricts movement and colonization of X. fastidiosa, may be an adaptation to endophytic growth of the pathogen that prevents the excessive growth of cells in vessels.

Download Full-text

AcpA Is a Francisella Acid Phosphatase That Affects Intramacrophage Survival and Virulence

Infection and Immunity ◽

10.1128/iai.01226-06 ◽

2006 ◽

Vol 75 (1) ◽

pp. 390-396 ◽

Cited By ~ 48

Author(s):

Nrusingh P. Mohapatra ◽

Ashwin Balagopal ◽

Shilpa Soni ◽

Larry S. Schlesinger ◽

John S. Gunn

Keyword(s):

Acid Phosphatase ◽

Intracellular Trafficking ◽

Type Strain ◽

Wild Type Strain ◽

Human Monocyte ◽

Human Macrophage ◽

Wild Type ◽

Intracellular Killing ◽

Francisella Novicida ◽

Transmission Electron

ABSTRACT AcpA of Francisella spp. is a respiratory-burst-inhibiting acid phosphatase that also exhibits phospholipase C activity. To better understand the molecular basis of AcpA in virulence, a deletion of acpA was constructed in Francisella novicida. The phosphatase and lipase activities were reduced 10-fold and 8-fold, respectively, in the acpA mutant compared to the wild type and were found mostly associated with the outer membrane. The acpA mutant was more susceptible to intracellular killing than the wild-type strain in the THP-1 human macrophage-like cell line. In addition, mice infected with the acpA mutant survived longer than the wild-type strain and were less fit than the wild-type strain in competition infection assays. Transmission electron microscopy showed that the acpA mutant was delayed in escape from macrophage phagosomes, as more than 75% of acpA mutant bacteria could still be found inside phagosomes after 12 h of infection in THP-1 cells and human monocyte-derived macrophages, whereas most of the wild-type bacteria had escaped from the phagosome by 6 h postinfection. Thus, AcpA affects intracellular trafficking and the fate of Francisella within host macrophages.

Download Full-text

Role of the luxS Gene in Initial Biofilm Formation by Streptococcus mutans

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000371816 ◽

2015 ◽

Vol 25 (1) ◽

pp. 60-68 ◽

Cited By ~ 24

Author(s):

Zhiyan He ◽

Jingping Liang ◽

Zisheng Tang ◽

Rui Ma ◽

Huasong Peng ◽

...

Keyword(s):

Streptococcus Mutans ◽

Mutant Strain ◽

Biofilm Formation ◽

Type Strain ◽

Laser Scanning ◽

Wild Type Strain ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Wild Type ◽

Initial Biofilm

Quorum sensing (QS) is a process by which bacteria communicate with each other by secreting chemical signals called autoinducers (AIs). Among Gram-negative and Gram-positive bacteria, AI-2 synthesized by the LuxS enzyme is widespread. The aim of this study was to evaluate the effect of QS luxS gene on initial biofilm formation by Streptococcus mutans. The bacterial cell surface properties, including cell hydrophobicity (bacterial adherence to hydrocarbons) and aggregation, which are important for initial adherence during biofilm development, were investigated. The biofilm adhesion assay was evaluated by the MTT method. The structures of the 5-hour biofilms were observed by using confocal laser scanning microscopy, and QS-related gene expressions were investigated by real-time PCR. The luxS mutant strain exhibited higher biofilm adherence and aggregation, but lower hydrophobicity than the wild-type strain. The confocal laser scanning microscopy images revealed that the wild-type strain tended to form smaller aggregates with uniform distribution, whereas the luxS mutant strain aggregated into distinct clusters easily discernible in the generated biofilm. Most of the genes examined were downregulated in the biofilms formed by the luxS mutant strain, except the gtfB gene. QS luxS gene can affect the initial biofilm formation by S. mutans.

Download Full-text

Consequences of Reduction of Klebsiella pneumoniae Capsule Expression on Interactions of This Bacterium with Epithelial Cells

Infection and Immunity ◽

10.1128/iai.67.2.554-561.1999 ◽

1999 ◽

Vol 67 (2) ◽

pp. 554-561 ◽

Cited By ~ 101

Author(s):

Sabine Favre-Bonte ◽

Bernard Joly ◽

Christiane Forestier

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Klebsiella Pneumoniae ◽

Cell Lines ◽

Type Strain ◽

Wild Type Strain ◽

Conjugative Plasmid ◽

Intestinal Cell ◽

Wild Type ◽

Defective Mutant

ABSTRACT Most Klebsiella pneumoniae clinical isolates are fully encapsulated and adhere in vitro to intestinal cell lines with an aggregative pattern. In this study, the influence of the capsule on interactions with epithelial cells was investigated by creating an isogenic mutant defective in the synthesis of the capsule. Determination of the uronic acid content of bacterial extracts confirmed that the mutant did not produce capsular polysaccharides whereas, with the wild-type strain, the level of encapsulation was growth phase dependent and reached a maximum during the lag and early log phases. Assays performed with different epithelial cell lines, Int-407, A-549, and HEp-2, showed that the capsule-defective mutant demonstrated greater adhesion than did the wild-type strain and that the aggregative pattern was maintained, indicating that the capsule was not related to the adhesion phenotype. In contrast, when the mucus-producing HT-29-MTX cells were used, the encapsulated wild-type strain adhered more strongly than did the capsule-defective mutant. No invasion properties were observed with any of the capsular phenotypes or cell lines used. The K. pneumoniae adhesin CF29K was detected by Western blot analysis and enzyme-linked immunosorbent assay on the surface of transconjugants obtained after transfer of a conjugative plasmid harboring the CF29K-encoding genes into both the wild-type and the capsule-defective strains. The amounts of adhesin detected were greater in the capsule-defective background strain than in the wild-type encapsulated strain and were associated with an increase in the level of adhesion to Caco-2 cells. Moreover, RNA slot blot experiments showed that transcription of the adhesin-encoding gene was markedly increased in the capsule-defective mutant compared to the wild-type encapsulated background. These results suggest (i) that the capsule plays an active role during the initial steps of the pathogenesis by interacting with mucus-producing cells but is subsequently not required for the adhesin-related interaction with the epithelial cell surface and (ii) that the expression of the adhesin is modulated by the presence of a capsule at a transcriptional level.

Download Full-text

Protein Arginine N-methyltransferases 5 and 7 Promote HIV-1 Production

Viruses ◽

10.3390/v12030355 ◽

2020 ◽

Vol 12 (3) ◽

pp. 355 ◽

Cited By ~ 3

Author(s):

Hironobu Murakami ◽

Takehiro Suzuki ◽

Kiyoto Tsuchiya ◽

Hiroyuki Gatanaga ◽

Manabu Taura ◽

...

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Virus Production ◽

Human Monocyte ◽

Wild Type ◽

Viral Reservoirs ◽

Immunodeficiency Virus ◽

Current Therapies ◽

Hiv 1

Current therapies for human immunodeficiency virus type 1 (HIV-1) do not completely eliminate viral reservoirs in cells, such as macrophages. The HIV-1 accessory protein viral protein R (Vpr) promotes virus production in macrophages, and the maintenance of Vpr is essential for HIV-1 replication in these reservoir cells. We identified two novel Vpr-binding proteins, i.e., protein arginine N-methyltransferases (PRMTs) 5 and 7, using human monocyte-derived macrophages (MDMs). Both proteins found to be important for prevention of Vpr degradation by the proteasome; in the context of PRMT5 and PRMT7 knockdowns, degradation of Vpr could be prevented using a proteasome inhibitor. In MDMs infected with a wild-type strain, knockdown of PRMT5/PRMT7 and low expression of PRMT5 resulted in inefficient virus production like Vpr-deficient strain infections. Thus, our findings suggest that PRMT5 and PRMT7 support HIV-1 replication via maintenance of Vpr protein stability.

Download Full-text