Abstract
Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of ‘ Candidatus Liberibacter asiaticus’ (Las), and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome and causes huanglongbing (HLB), a devastating disease of citrus worldwide. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of a typical signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria when expressed in the plants. These findings may lead to a better understanding of the pathogenesis of this intracellular bacterium.
Highlights
Autotransporters are large multi-domain virulence factors encoded by genomes of diverse gram-negative bacteria
Using the SignalP signal peptide prediction software, no signal sequence was identified in LasAI or LasAII and little information about the function of LasAI or LasAII was obtained from a BLAST search of the NCBI protein database
Despite the absence of typical signal peptides and no significant sequence homology with other autotransporters at the amino acid level, sequence analyses predicted that LasAI and LasAII possess architectural features of the autotransporter family, including passenger domains with large repeated sequences that form coiled-coils and translocator domains containing β-stranded structures
Summary
Autotransporters are large multi-domain virulence factors encoded by genomes of diverse gram-negative bacteria. A typical autotransporter consists of three functional domains: a Sec-dependent N-terminal signal peptide, a secreted passenger domain (α-domain) and a conserved C-terminal translocator domain (β-domain) [1]. The central passenger domain will be either attached to the cell surface or secreted. This type of self-transporting protein system is referred to as a type V secretion system (T5SS). Known virulence factors secreted by T5SS have been shown to be cytotoxic, contain protease activities, or functions such as adhesions. The trimeric autotransporters (T5cSS) known as AT-2 are exemplified by the oligomeric coiled-coil adhesions from various pathogenic bacteria, such as YadA of Yersinia [5], Hia of Haemophilus [6], and Hap of Haemophilus [7]. It was demonstrated that the YadA translocator localized solely to the mitochondrial outer membrane when expressed in yeast and that four βstands are sufficient for mitochondrial localization [12]
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