OHSU

Cambronne Lab

Overview

Pathogenic bacteria have evolved specialized secretion systems dedicated to the injection of proteins directly into target eukaryotic host cells. Once in the host cell cytoplasm, these ‘effector’ proteins manipulate cellular processes to create a favorable environment for survival and replication of the bacterium. The type IV secretion pathway is employed by numerous important Gram-negative bacterial pathogens and thus represents a primary virulence determinant in many human diseases. While this secretion pathway has emerged as a conserved virulence strategy, the mechanisms governing effector protein recognition and transport remain poorly defined. L.pneumophila is a facultative intracellular pathogen that can cause a severe pneumonia in humans called Legionnaires disease. This bacterium employs the Dot/Icm transporter to deliver at least 150 effector proteins of diverse molecular character into eukaryotic host cells. Our objectives intend to expand our understanding of this translocation pathway using genetic, molecular and biochemical techniques in order to elucidate the type IV secretion mechanisms that promote effector protein translocation.

In detail

Translocation of effector proteins by the Dot/Icm T4SS. Effector proteins (yellow) are recognized and delivered to the host cell cytoplasm by an unknown mechanism that requires 26 dot/icm gene products (blue).

Type IV secretion determinants in Legionella pneumophila

The type IV secretion system (T4SS) is a collective term assigned to multi-protein complexes that promote the transport of bacterial-derived proteins or protein/nucleic acid hybrids across the envelope of the bacterial cell. Translocation of effector proteins into a eukaryotic cell can be mediated by a specialized T4SS, and several human pathogens require a T4SS for virulence. Legionella pneumophila, the causative agent of Legionnaires’ disease, requires the Dot/Icm T4SS for intracellular replication in alveolar macrophages. Although the apparatus constituting a functional T4SS may be comprised by 10-12 different proteins, the Dot/Icm T4SS in L. pneumophila is composed of at least 26 different proteins (Fig.1). Additionally, the Dot/Icm transporter is predicted to deliver as many as 150 effector proteins to the host cell cytoplasm. Thus, the Dot/Icm T4SS represents one of the most complex secretion systems in the prokaryotic kingdom. Our research is focused on determining the molecular mechanisms that govern substrate selection and protein translocation by the Dot/Icm transporter. Many of the essential Dot/Icm components are integral membrane proteins; however some Dot/Icm proteins are localized to the bacterial cytoplasm. Genetic studies indicated that two of the soluble factors, IcmS and IcmW were dispensable for assembly of the competent Dot/Icm apparatus, yet remained critical for intracellular replication of L. pneumophila in host phagocytes. This observation prompted a model where the IcmS and IcmW proteins could represent components necessary for the selection of a particular class of effector proteins. Our current research has focused on the biochemical contribution of IcmS and IcmW in effector protein translocation.


Model for contribution of IcmSW in Dot/ICM-mediated translocation. IcmSW binds to effector proteins in a region distinct from the C-terminal translocation signal to promote recognition by the T4SS.

Studies have indicated that IcmS and IcmW form a complex in the L. pneumophila cytoplasm, and we have demonstrated that the IcmSW complex is necessary for the translocation of numerous effector proteins of diverse molecular character. Biochemical experiments have revealed that the IcmSW complex binds to a central region in the effector protein to promote/support a conformation in the substrate that facilitates recognition of the C-terminal translocation signal found among numerous T4SS effectors (Fig.2). Thus, our studies indicate that the IcmSW complex defines a chaperone system that promotes recognition of effector proteins by the T4SS apparatus by maintaining substrates in a translocation competent state. This work provides important molecular details of an initial stage in protein translocation by the Dot/Icm system. Our future work will focus on new questions raised by studies on the IcmSW complex, emphasizing mechanisms that govern effector protein recognition and translocation. Further, effector proteins have diverse molecular properties that present additional complexities that must be overcome for efficient protein translocation.