Metagenome diversity illuminates the origins of pathogen effectors
Authors: Lehman Stephanie S. , Verhoeve Victoria I. , Driscoll Timothy P. , Beckmann John F. , Gillespie Joseph J. 
Published: April 2024

Recent metagenome-assembled genome (MAG) analyses have profoundly impacted Rickettsiology systematics. The discovery of basal lineages (novel families Mitibacteraceae and Athabascaceae) with predicted extracellular lifestyles exposed an evolutionary timepoint for the transition to host dependency, which seemingly occurred independent of mitochondrial evolution. Notably, these basal rickettsiae carry the Rickettsiales
vir
homolog (
rvh
) type IV secretion system and purportedly use
rvh
to kill congener microbes rather than parasitize host cells as described for later-evolving rickettsial pathogens. MAG analysis also substantially increased diversity for the genus
Rickettsia
and delineated a sister lineage (the novel genus
Tisiphia
) that stands to inform on the emergence of human pathogens from protist and invertebrate endosymbionts. Herein, we probed Rickettsiales MAG and genomic diversity for the distribution of
Rickettsia rvh
effectors to ascertain their origins. A sparse distribution of most
Rickettsia rvh
effectors outside of Rickettsiaceae lineages illuminates unique
rvh
evolution from basal extracellular species and other rickettsial families. Remarkably, nearly every effector was found in multiple divergent forms with variable architectures, indicating profound roles for gene duplication and recombination in shaping effector repertoires in
Rickettsia
pathogens. Lateral gene transfer plays a prominent role in shaping the
rvh
effector landscape, as evinced by the discovery of many effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchange between
Rickettsia
and
Legionella
species. Our study exemplifies how MAGs can yield insight into pathogen effector origins, particularly how effector architectures might become tailored to the discrete host cell functions of different eukaryotic hosts.

IMPORTANCE

While rickettsioses are deadly vector-borne human diseases, factors distinguishing
Rickettsia
pathogens from the innumerable bevy of environmental rickettsial endosymbionts remain lacking. Recent metagenome-assembled genome (MAG) studies revealed evolutionary timepoints for rickettsial transitions to host dependency. The
rvh
type IV secretion system was likely repurposed from congener killing in basal extracellular species to parasitizing host cells in later-evolving pathogens. Our analysis of MAG diversity for over two dozen
rvh
effectors unearthed their presence in some non-pathogens. However, most effectors were found in multiple divergent forms with variable architectures, indicating gene duplication and recombination-fashioned effector repertoires of
Rickettsia
pathogens. Lateral gene transfer substantially shaped pathogen effector arsenals, evinced by the discovery of effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchanges between
Rickettsia
and
Legionella
species. Our study exemplifies how MAGs yield insight into pathogen effector origins and evolutionary processes tailoring effectors to eukaryotic host cell biology.