Intracellular pathogens form intimate relationships with host cells to establish niches that support optimal growth. To maintain this ideal environment, pathogens combat host defenses such as inflammatory responses, genotoxicity, and cell death. One such host cell guardian is the tumor-suppressing transcription factor, p53, which controls the host response to DNA damage to maintain cellular homeostasis and ultimately provide balance on the multicellular, or host, level. Mutations in the gene that encodes p53, TP53, are a hallmark of more than half of human cancers including those caused by infections like human papillomavirus (HPV). The importance of p53 to host cell homeostasis makes it a major target of pathogens.
Scrub typhus is a disease with over a million cases annually in the Asia-Pacific and non-travel related cases in Africa, South America, and the Middle East. The causative agent, O. tsutsugamushi, is an obligate intracellular bacterium that has been found on every continent except Antarctica. Scrub typhus can cause a range of symptoms from mild fever and chills to major organ failure and death. Effective therapeutics against this disease are limited and there is no vaccine available. Despite the clear indication that scrub typhus is a global health threat, our understanding of Orientia pathobiology and how it leads to disease is lacking.
We initiated our study to shed light on host components fundamental for O. tsutsugamushi pathogenesis. We began by assessing changes in host gene expression using RNAseq. Orientia has a replication cycle spanning 3-5 days. So, in attempts to fully capture alterations in host dynamics during infection we defined the host transcriptome at different timepoints across the cycle. This analysis revealed three overarching themes: 1) The host transcriptome is not majorly affected until the O. tsutsugamushi burden is high; 2) antimicrobial transcriptional responses (e.g., interferon, NF-kB, IL-10, inflammasome activation) are upregulated at a high O. tsutsugamushi load; 3) aside from antimicrobial responses, TP53 and genes involved in its downstream pathways (e.g., proliferation and the cell cycle, DNA damage response, and apoptosis) are the only transcripts altered across O. tsutsugamushi’s replication cycle. The first two patterns are consistent with previous work investigating intracellular pathogens, especially those that must persist in host cells for an extended timeframe, like O. tsutsugamushi. However, the third finding was unexpected. Because p53 is a master regulator of host cell integrity and therefore necessary for preservation of a promicrobial intracellular environment, we rationalized that dysregulation of p53 and its pathways could be essential for O. tsutsugamushi niche optimization.
TP53 is significantly downregulated in Orientia infected cells, and this translates to lower p53 protein levels. Many intracellular pathogens like HPV, Chlamydia trachomatis, and Plasmodium yoelii predominantly target p53 for degradation by the 26S proteasome by coopting host cell ubiquitination machinery. As O. tsutsugamushi also hijacks this machinery for its benefit, the next logical step was to determine if Orientia sends p53 to the proteasome. Intriguingly, O. tsutsugamushi requires neither host ubiquitination machinery nor the proteasome to rid the cell of p53 and thus only targets TP53 expression, which has yet to be shown for any other microbe.
p53 controls several different host cell processes vital to homeostasis. We assessed if O. tsutsugamushi infection has an impact on these pathways including host cell proliferation and the cell cycle, DNA damage repair responses, and apoptosis. p53 inhibits host proliferation and the cell cycle at various checkpoints, thus we expected that proliferation would be uncontrolled during Orientia infection. However, we observed the opposite. O. tsutsugamushi delays host cell proliferation in the DNA replication phase of the cell cycle for reasons that are currently unclear. Next, we determined if the DNA damage repair response is altered during infection. Interestingly, infected cells do not exhibit DNA damage or mount a repair response until the bacterial burden is high. Moreover, Orientia inhibits genotoxicity in the presence of the DNA damaging anti-cancer therapeutic, etoposide. In congruence with these data, O. tsutsugamushi also inhibits apoptosis even when DNA damage is induced until late infection. These findings are two-fold: 1) O. tsutsugamushi impedes TP53 expression and prohibits genotoxicity and programmed cell death; 2) the lack of p53 convecably allows Orientia to fine-tune critical host processes, like the host cell cycle, to protect its niche and support replication. O. tsutsugamushi protein expression is necessary to get rid of p53. We presumed that a bacterial effector protein is involved. Fortunately, we had previously identified an ankyrin repeat-containing effector, Ank13, that globally modulates host cell gene expression including TP53. We confirmed that Ank13 reduces TP53 and p53 as well as protects against genotoxicity and apoptosis.
Intracellular pathogens have developed strategies to remain incognito while establishing and maintaining an ideal environment for microbial replication. Because p53 is a major regulator of numerous pathways essential for host cell survival, it is unmistakably an important contributor to microbial disguise mechanisms and ultimately pathogenesis. Overall, this study highlights p53 as a key target for O. tsutsugamushi and possibly other pathogens to maintain the intracellular niche and suggests that p53 dysregulation creates a malleable environment that microbes hone to meet their specific fitness needs. Finally, our findings further validate p53 as an anti-microbial molecule and with the vast cohort of anti-cancer therapeutics aimed to restore p53 levels, provides an avenue for use of already developed host-directed drugs against intracellular pathogens.
Poster image generated by Paige Allen, Ph.D. and Tytus Bernas, Ph.D.