Manipulation and survival: insights into strategies of the facultative intracellular pathogen "Brucella"

About 130 years ago the first microbe was discovered. Since then, we made remarkable progress in understanding the governing factors of host-pathogen relationships. We now understand that bacteria evolved a copious amount of virulence factors to manipulate a plethora of host cell functions in their favor to establish successful infections, but also that a multitude of phenotypic adaptations ensure their survival in the presence of different stressors. However, even though we begin to understand the intricate interactions between the facultative intracellular zoonotic pathogen Brucella and its hosts we still lack a holistic view on host as well as bacterial factors governing the pathogen-host interactions of this intriguing pathogen.
As an intracellular pathogen Brucella evolved distinct mechanisms to engineer its intracellular trafficking to subvert destruction in a phagolysosomal compartment by interaction with different organelles of the host cell. In Research article I (published in mSphere) we employed a genome-wide RNA interference screen in combination with Brucella infection to discover new host cell factors involved in intermediate trafficking stages. Using this approach, we discovered that the retromer, a part of the endosomal protein sorting machinery, is involved in Brucella intracellular trafficking. We show that knockdown of VPS35, a core component of the retromer, resulted in arrest of the trafficking at the late endosomal stage and therefore hinders the transition of the endosomal Brucella-containing vacuole to the replication submissive ER-associated vacuole. Our results therefore implicate that the host cell’s retromer complex plays a vital role in Brucella intracellular trafficking.
To fight infections caused by bacterial pathogens humans have developed different therapies. Antibiotic therapy for Brucellosis is long and burdensome and still results in a relatively high relapse rate of 5-15%, even though occurrence of stable genetic resistance is comparably rare. Poor compliance and reinfection likely account for some relapse cases. In Research article II (published in PLoS Neglected Tropical Diseases) we identified another phenomenon likely contributing to the high relapse rate by utilization of Brucella carrying a dual reporter for constitutive vs induced gene expression in combination with in vitro and in cellulo experiments. We quantified the protective nature of Brucella’s intracellular niche(s) and established that the intracellular lifestyle decreases antibiotic efficacy. Brucella is able to form a subpopulation of transiently non-growing phenotypically antibiotic tolerant bacteria, which can resume metabolic activity and remain fully virulent after discontinuation of antibiotic treatment in a cellular model. This subpopulation of antibiotic persisters therefore constitutes a possible reservoir for relapses and should be addressed in adapted anti-Brucellosis strategies. To ensure intracellular survival Brucella utilizes translocated effector proteins to orchestrate host cell functions. Brucella further has to adapt to the changing intracellular environments to withstand a plethora of different stresses. Brucella’s Type 4 Secretion System (T4SS) was coined the main contributor to effector translocation into host cells, but different effector proteins were shown to be translocated by T4SS-independent and yet unknown mechanisms corroborating that other mechanisms are involved in introducing virulence factors to the host cell. In Research article III (published in mSphere ) we show that the putative T4SS effector (T4SSE) BspD is a signature protein of the Rhizobiales involved in stress resistance and intracellular survival and/or replication of Brucella therefore constituting a fitness determinant of Brucella. In Research article IV (in preparation) we present data showing that a subset of putative T4SSEs harbor functional signal peptides leading to export to the periplasm via the SecYEG translocon and that these proteins are secreted from the bacterial cell with OMVs in a T4SS-independent manner in vitro. We therefore propose that OMVs could play a significant role in pathogen-host interaction of Brucella which should be further investigated. Another aspect of this study was the establishment of a biosafety level 2 compatible Brucella model using Brucella microti (B. microti) with a suspected low zoonotic potential to enhance research output and simplify experimentation in comparison to a biosafety level 3 set-up using Brucella abortus (B. abortus). We therefore corroborated published findings that B. microti displays faster replication and replicates in a T4SS-dependent manner in mouse macrophages. We further validated that B. microti replicates in an ER-derived compartment as observed for classical Brucella species such as B. abortus.
Together these results contribute to the formation of a more holistic understanding of infection and survival strategies of the facultative intracellular pathogen Brucella.