The virB-encoded type IV secretion system is critical for establishment of infection and persistence of Brucella ovis infection in mice

Abstract

Brucella spp. are gram-negative intracellular bacterial pathogens that cause chronic infections. Brucella virulence factors include a type IV secretion system (T4SS) and its lipopolysaccharide (LPS), which are essential for persistence. However, the role of the virB-encoded T4SS has not been investigated in naturally rough Brucella species such as Brucella ovis. In this study, male 6-week old BALBc mice were infected with B. ovis, Brucella abortus, and their respective ΔvirB2 mutant strains. During early infection, B. ovis and B. abortus wild type strains were similarly recovered from spleen. Interestingly, in contrast to ΔvirB2 B. abortus that was recovered at similar levels when compared to the wild type strain, the ΔvirB2 B. ovis was markedly attenuated as early as 24 h post infection (hpi). The ΔvirB2 B. ovis was unable to survive and multiply in murine peritoneal macrophages and extracellularly within the peritoneal cavity at 12 and 24 hpi with lower splenic colonization than the parental strain at 6, 12 and 24 hpi. In contrast, wild type B. abortus and ΔvirB2 B. abortus had a similar kinetics of infection in this model. As expected, the T4SS was essential for intracellular replication of smooth and rough strains in RAW macrophages at 48 hpi. These results suggest that T4SS is important for survival of B. ovis in murine model, and that a T4SS deficient B. ovis strain is cleared at earlier stages of infection when compared to a similar B. abortus mutant.

Introduction

Brucella spp. are gram-negative intracellular alpha-proteobacteria pathogens (Garrity, 2001). Brucellosis is a zoonotic disease with variable clinical manifestations (Xavier et al., 2009). In humans, brucellosis is characterized by intermittent fever, anorexia, weakness, and chronic inflammation in several organs as liver, spleen, heart, bone and brain (Young, 1995). In animals, Brucella causes reproductive failure associated with abortions, orchitis and epididymitis. Brucella ovis infects sheep, causing primarily epididymitis and occasionally abortion. It has not been associated with human infections (Xavier et al., 2009).

Brucella virulence is associated with its ability to survive intracellularly (Celli et al., 2003, Billard et al., 2005, Carvalho Neta et al., 2008). Most Brucella species have smooth LPS, whereas Brucella canis and B. ovis have a naturally rough LPS due to the lack of the O-chain. Smooth Brucella LPS plays a role in resistance to complement and cationic antimicrobials (Martinez de Tejada et al., 1995, Fernandez-Prada et al., 2003, Lapaque et al., 2005). Therefore, mutant rough strains of Brucella abortus, B. melitensis or B. suis are attenuated (Allen et al., 1998, Jimenéz de Bagüés et al., 2004).

The virB operon-encoded type IV secretion system (T4SS) is essential for virulence of smooth Brucella species (O’Callaghan et al., 1999, Hong et al., 2000, Delrue et al., 2001, Roux et al., 2007). virB mutant strains of B. melitensis, B. abortus, and B. suis are attenuated (O’Callaghan et al., 1999, Hong et al., 2000, Den Hartigh et al., 2004, Rolán and Tsolis, 2007, Paixão et al., 2009, Zhong et al., 2009, Wang et al., 2010). Brucella strains with a functional T4SS can evade degradation in lysosomes, and are able to fuse with the rough endoplasmatic reticulum (Celli et al., 2003). The role of the virB-encoded T4SS in naturally rough Brucella species has not yet been investigated. In this study the role of T4SS in B. ovis infection is evaluated.