Interaction between mammalian cells and Pasteurella multocida B:2. Adherence, invasion and intracellular survival

doi:10.1016/j.micpath.2012.03.005

Microbial Pathogenesis

Volume 52, Issue 6, June 2012, Pages 353-358

https://doi.org/10.1016/j.micpath.2012.03.005 Get rights and content

Abstract

A Pasteurella multocida B:2 strain from a case of bovine haemorrhagic septicaemia (HS) and a derivative, JRMT12, that was attenuated by a deletion in the aroA gene, were shown to adhere to, invade and survive within cultured embryonic bovine lung (EBL) cells. By comparison, bovine strains of Mannheimia haemolytica serotype A1 and P. multocida serotype A:3, although able to adhere to EBL cells, were not found intracellularly. The B:2 strains were viable intracellularly over a 7 h period, although a steady decline in viability was noted with time. Entry into the mammalian cells was inhibited by cytochalasin D, indicating that cell uptake was by an actin-dependent process. Viability assessment of EBL cells by trypan blue staining indicated that none of the bacterial strains was toxic for the EBL cells. Transmission electron microscopy (TEM) showed that, after entry into the mammalian cells, the B:2 strain resided in a vacuolar compartment. However, only a low percentage of mammalian cells appeared to contain one or more P. multocida B:2, suggesting that only certain EBL cells in the population were capable of being invaded by, or of taking up, the bacteria. TEM showed that P. multocida A:3 and M. haemolytica A:1 were found loosely adhering to the cell surface of EBL cells and were not detected intracellularly. The cell-invasive capacity of P. multocida B:2 may be a virulence property related to its ability to translocate from the respiratory tract into the blood stream.

Highlights

► Bovine Pasteurella multocida and Mannheimia haemolytica strains adhered to embryonic bovine lung (EBL) cells. ► Only P. multocida B:2 strains were found to invade the EBL cells. ► One strain was found to survive intracellularly in EBL cells for up to 7 h. ► Bacterial entry into mammalian cells appeared to be by an actin-dependent process.

Introduction

Pasteurella multocida B:2 causes haemorrhagic septicaemia (HS) in cattle and buffaloes [1]. The successful experimental transmission of disease by the intranasal and oral routes, producing a syndrome with clinical signs and lesions resembling natural disease, indicates that these may be the natural routes of infection [2]. However, a feature of HS disease is the rapid spread of infecting bacteria from the respiratory tract to the blood and lymph to cause a fatal septicaemia in less than 48 h. To pass into the bloodstream, the bacteria must migrate through the epithelial layer into the pulmonary interstitium. A potential of Pasteurella B:2 for attachment to and invasion of mammalian cells may constitute a mechanism that enables the bacteria to invade the bloodstream. In this study, two strains of P. multocida B:2 were assessed for their ability to adhere to and invade embryonic bovine lung (EBL) cells. For comparison, bovine strains of P. multocida A:3 and Mannheimia haemolytica A:1 were included.

Section snippets

Effect of P. multocida B:2 on viability of EBL cells

To determine the effect of P. multocida B:2 on the viability of EBL cells, the cells were trypsinized at 3 h post-infection with bacteria at MOI 100:1, suspended in EBL assay medium and stained with trypan blue (section 4.3). Bovine strains of P. multocida A3 and M. haemolytica A:1 were also tested. EBL viability in the presence of bacteria was compared to that of the control cells where no bacteria were present. None of the bacterial strains showed a toxic effect towards EBL viability during

Discussion

In this study, the attenuated derivative P. multocida B:2 JRMT12 adhered to EBL cells better than its parent strain, 85020. This might possibly be due to modification in the surface properties associated with altered metabolism as a result of disruption of aromatic acid biosynthesis in the JRMT12 strain. The better adherence of JRMT12 compare to the wild-type strain may explain the slightly better invasive capacity of the mutant. The other strains, P. multocida A:3 and M. haemolytica A:1

Bacterial strains and growth conditions

Bacterial strains used in this study were: a bovine isolate of P. multocida B:2 85020, originally isolated from a case of HS in Sri Lanka; P. multocida B:2 JRMT12, an ΔaroA mutant of strain 85020 and potential vaccine strain [9]; a bovine isolate of P. multocida A:3 [10]; and a bovine isolate of M. haemolytica A:1 [11]. Both P. multocida and M. haemolytica strains were grown on Brain Heart Infusion (BHI) agar and in BHI broth (Difco) media at 37 °C and shaken at 180 rpm.

Preparation of embryonic bovine lung (EBL) cells

EBL cells (German

Acknowledgements

Sarah Othman was supported by the Ministry of Higher Education Malaysia and Universiti Putra Malaysia.

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High- and low-virulent bovine Pasteurella multocida induced differential NLRP3 inflammasome activation and subsequent IL-1β secretion
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P. multocida can invade various cell types, including chicken embryo fibroblast cells, turkey kidney epithelial cells, bovine endothelial cells, etc. (Al-haj Ali et al., 2004; Galdiero et al., 2001; Lee et al., 1994). P. multocida can enter into macrophages and this is important for the induction of immune responses (Othman et al., 2012; Puspitasari et al., 2019; Wilkie et al., 2012). We have previously reported that NLRP3 inflammasome, but not AIM2 or NLRC4 inflamamsome, is assembled in macrophages upon PmCQ2 infection (Fang et al., 2019).
Pasteurella multocida is a gram-negative bacterial pathogen, which causes a large number of diseases in mammals, birds and human. Although the bacterium has been known for decades, the pathogenesis and the mechanisms of P. multocida induced host immunity are poorly understood. Recently, we have reported that nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome plays an important role in caspase-1 activation and IL-1β secretion in macrophages infected with P. multocida. In this study, the inflammasome activation and IL-1β secretion were further demonstrated by using high- and low-virulent bovine P. multocida isolates. The results showed that, comparing with macrophages infected with the high-virulent PmCQ2 isolates, the low-virulent PmCQ6 induced higher levels of NLRP3 transcription, caspase-1 activation and mature IL-1β secretion. Furthermore, the capsule of the high-virulent PmCQ2 was much thicker than that of low-virulent PmCQ6, which indicating that capsular thickness might influence the bacteria colonization and NLRP3 inflammasome activation. The results suggested that differences in maturation of IL-1β in macrophages upon high- and low- virulent P. multocida infection are critically dependent on the differential activation of NLRP3 inflammasome. This study provided more understanding for the host immune responses induced by P. multocida and further extended the knowledge of P. multocida virulence from the view of host innate immunity.
In-vitro phagocytosis and intracellular killing of Pasteurella multocida B:2 by phagocytic cells of buffaloes
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Citation Excerpt :
Rapid spread of the infecting bacterium from the respiratory tract to the blood and lymph leads to fatal septicaemia within 16–72 h [6]. The bacterium migrates through the epithelial layer into the pulmonary interstitium leading to septicaemia [7]. As the first line of defense, both neutrophils and macrophages play important role in phagocytosis and killing of invading organisms.
Pasteurella multocida B:2 is a Gram-negative organism causing haemorrhagic septicaemia (HS) in buffaloes. It causes severe pulmonary infection, leading to infiltration of numerous macrophages and neutrophils. Despite the inflammatory response, buffaloes succumb to HS. This study aims to evaluate the in-vitro efficacy of macrophages and neutrophils of buffalo following exposure to P. multocida B:2. In-vitro infections were done using 10⁷ cfu/ml of P. multocida B:2 for Group 1, Escherichia coli for Group 2 and Mannhaemia haemolytica A:2 for Group 3 cells. The inoculated cell cultures were harvested at 0, 30, 60 and 120 min post-exposure and the phagocytic, killing and cell death rates were determined. Both phagocytosis and killing rates of all bacteria increased over time. Phagocytosis involved between 71% and 73% neutrophils and between 60% and 64% macrophages at 120 min. Killing rate of all bacteria involved between 76% and 79% for neutrophils and between 70% and 74% for macrophages at 120 min. Death rate of neutrophils ranged between 67% in Group 3, and 88% in Group 1 at 120 min, significantly (p < 0.05) higher than Group 3 but insignificant (p > 0.05) than Group 2. Similar pattern was observed for death rate of macrophages. The phagocytosis and killing rates of P. multocida B:2 were similar to other bacterial species used in this study but more neutrophils and macrophages were dead following infection by P. multocida B:2 than M. haemolytica A:2.
NLRP3 inflammasome plays an important role in caspase-1 activation and IL-1β secretion in macrophages infected with Pasteurella multocida
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However, in our study, we showed that the phagocytosis of P. multocida was required for IL-1β secretion; phagocytosis of bacteria may be required for NLRP3 inflammasome activation. Other studies have provided evidence that the entry of P. multocida into macrophages is important for the induction of the immune response (Othman et al., 2012; Wilkie et al., 2012). We have developed a mechanistic model of caspase-1 activation and IL-1β maturation in macrophages infected by P. multocida.
Pasteurella multocida is a Gram-negative bacterium that is responsible for a variety of diseases in birds and mammals, including humans. We have previously reported that the P. multocida serotype A strain PmCQ2 causes severe lung pneumonia in bovines. Transcriptomic analysis showed that many genes related to the immune response were significantly upregulated in the lungs of mice infected with P. multocida compared with uninfected mice. However, the mechanism by which P. multocida induces host inflammatory cytokine secretion is poorly understood. In this study, the mechanism of caspase-1 activation and subsequent IL-1β secretion in macrophages infected with P. multocida was elucidated. The nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome was shown to be involved in inducing this cellular response. Compared with wild-type macrophages, Nlrp3^−/− macrophages exhibited a clear decrease in caspase-1 activation and IL-1β secretion in response to P. multocida infection. Furthermore, spleen tyrosine kinase (Syk) was indicated to be involved in IL-1β secretion, possibly by regulating the NLRP3 inflammasome. Our results provide new insight into the host proinflammatory immune response against P. multocida and the critical involvement of the NLRP3 inflammasome in this activity.
The ABA392/pET30a protein of Pasteurella multocida provoked mucosal immunity against HS disease in a rat model
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Mucosal vaccination, is the most preferable defense for shielding the host from P. multocida B:2 infection in bovines, since this method reduces the susceptibility of bovines to the pathogen [11]. Transmission of disease of P. multocida B:2 bacteria is through intranasal and oral routes of bovines [12]. Thus, intranasal administration of mucosal protein vaccine is chosen since it mimics the oronasal route of infection and able to provoke both systemic and mucosal immunity.
Haemorrhagic septicaemia (HS) is a well-known high fatality septicaemic disease happening among bovines. The disease is caused by the Pasteurella multocida serotype B:2 bacteria. P. multocida B:2 has high mortality and morbidity rates and is spread through the intranasal and oral routes in bovines. In this study, our aim was to investigate the efficacy of the recombinant protein vaccine, ABA392/pET30a via intranasal inoculation by targeting the mucosal immunity. The constructed recombinant protein vaccine ABA392/pET30a was subjected to an animal study using Sprague Dawley rats. The study was divided into two parts: active and passive immunization studies. Both studies were carried out through the determination of immunogenicity (using Total White Blood Cell (TWBC) Count with Indirect ELISA) and histopathogenicity, analyzing (Bronchus Associated Lymphoid Tissue (BALT) formation) in lungs. As a result, the IgA and IgG development of both tested groups: group 1 (50μg/mL protein vaccine) and group 2 (100μg/mL protein vaccine) showed equivalent with the positive control group 4 (formalin-killed P. multocida B:2). However, there was a significant difference when compared with the negative control group 3 (normal saline). These results demonstrate that both the protein vaccine at the concentration 50μg/mL and 100μg/mL have the same efficacy as the commercially available positive control vaccine. From the studies, higher concentration of protein vaccine at 100μg/mL showed higher development of both IgA and IgG compared to 50μg/mL protein vaccine. Higher and rapid development of IgA compared to IgG showed that mucosal immunity has been induced through the intranasal administration of the protein vaccine. In addition, leucocytosis was observed at each dose of vaccination showed that the protein vaccine is capable to induce the immune responses of the host. Histopathogenicity studies of the vaccinated groups showed more BALT formation and no severe lesions after challenge compared to the negative control group. Besides, no inflammatory onsite or anaphylactic responses were observed after the intranasal inoculation which proved to be safer and provided longer lasting immunity. Therefore, recombinant protein vaccine ABA392/pET30a could be a potential candidate for intranasal administration which can provoke mucosal immunity against HS disease.
The ability of lipopolysaccharide (LPS) of Pasteurella multocida B:2 to induce clinical and pathological lesions in the nervous system of buffalo calves following experimental inoculation
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Factors such as type, virulence and dose of the bacteria, in addition to age and immune status of the animal are believed to play vital role in emergence of HS outbreaks [3,5–7]. Lipopolysaccharide (LPS), outer membrane proteins, capsule and frimbae are important virulence factors of P. multocida and play an essential role in the pathogenesis of HS [8–12]. Like other Gram negative bacteria, P. multocida LPS is a major component and considered as an essential virulence factor that plays an important role in the pathogenesis of the disease by protecting the bacterium from phagocytic activity and also accounts for most features of the clinical syndrome [11,13–16].
Lipopolysaccharide (LPS) of P. multocida B:2, a causative agent of haemorrhagic septicaemia (HS) in cattle and buffaloes, is considered as the main virulence factor and contribute in the pathogenesis of the disease. Recent studies provided evidences about the involvement of the nervous system in pathogenesis of HS. However, the role of P. multocida B:2 immunogens, especially the LPS is still uncovered. Therefore, this study was designed to investigate the role of P. multocida B:2 LPS to induce pathological changes in the nervous system. Nine eight-month-old, clinically healthy buffalo calves were used and distributed into three groups. Calves of Group 1 and 2 were inoculated orally and intravenously with 10 ml of LPS broth extract represent 1 × 10¹² cfu/ml of P. multocida B:2, respectively, while calves of Group 3 were inoculated orally with 10 ml of phosphate buffer saline as a control. Significant differences were found in the mean scores for clinical signs, post mortem and histopathological changes especially in Group 2, which mainly affect different anatomic regions of the nervous system, mainly the brain. On the other hand, lower scores have been recorded for clinical signs, gross and histopathological changes in Group 1. These results provide for the first time strong evidence about the ability of P. multocida B:2 LPS to cross the blood brain barrier and induce pathological changes in the nervous system of the affected buffalo calves.
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¹: Permanent address: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.

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Microbial Pathogenesis

Abstract

Highlights

Introduction

Section snippets

Effect of P. multocida B:2 on viability of EBL cells

Discussion

Bacterial strains and growth conditions

Preparation of embryonic bovine lung (EBL) cells

Acknowledgements

References (11)

Invasion of chicken embryo fibroblast cells by avian Pasteurella multocida

Vet Microbiol

Interactions between bovine endothelial cells and Pasteurella multocida: association and invasion

Res Microbiol

Two stages of enteropathogenic Escherichia coli intestinal pathogenicity are up and down-regulated by the epithelial cell differentiation

Res Bio Div

Vacuolating cytotoxic activity of Pasteurella multocida causing haemorrhagic septicaemia in buffalo and cattle

FEMS Microb Lett

Experimental induction of pneumonic pasteurellosis in calves by intratracheal infection with Pasteurella multocida biotype A:3

Res Vet Sci

Cited by (18)

High- and low-virulent bovine Pasteurella multocida induced differential NLRP3 inflammasome activation and subsequent IL-1β secretion

In-vitro phagocytosis and intracellular killing of Pasteurella multocida B:2 by phagocytic cells of buffaloes

NLRP3 inflammasome plays an important role in caspase-1 activation and IL-1β secretion in macrophages infected with Pasteurella multocida

The ABA392/pET30a protein of Pasteurella multocida provoked mucosal immunity against HS disease in a rat model

The ability of lipopolysaccharide (LPS) of Pasteurella multocida B:2 to induce clinical and pathological lesions in the nervous system of buffalo calves following experimental inoculation

Molecular characterisation of the GdhA- Derivative of pasteurella multocida B:2