(512) A PhoRP-regulated pilin in Mycobacterium abscessus is required for association with and survival in macrophages and CF mice

Thursday, November 3, 2022

12:00 PM – 12:40 PM ET

Presenting Author(s)

Matthew M. Schaefers, PhD (he/him/his)

Assistant Professor
Boston Children's Hospital
Boston, Massachusetts, United States

Co-Author(s)

MD

Marco DiBlasi

United States
KM

Kathryn Mansour

United States
GP

Gregory Priebe

Associate Professor
Boston Children's Hospital, United States

Background: Background. Infections caused by members of the Mycobacterium abscessus complex are a serious concern in people with cystic fibrosis (CF). These infections are often treatment-refractory as antibiotic resistance is common among members of the complex. The M. abscessus complex is composed of three species, M. abscessus, M. bolletii, and M. massiliense. The pathogenesis of M. abscessus complex infection remains poorly understood. Previous work has found that phoRP deletion mutants in both clinical CF isolates of M. abscessus and M. massiliense were less virulent than parental strains. Using RNA-seq we identified several putative phoRP-regulated virulence factors, including a pilin in M. abscessus. This pilin has a putative homolog in Mycobacterium tuberculosis whose role in virulence is unclear, but its function in M. abscessus is unknown. We hypothesize that this pilin contributes to phoRP-mediated virulence.

Methods: Methods. We constructed a pilin deletion mutant in a clinical isolates of M. abscessus (strain NTM635) and compared its ability to associate with and survive within THP-1-derived human macrophages. We also compared the virulence of these strains using a pneumonia model in transgenic CF mice (cftr F508 deletion mutant in the FVB/N background) using intranasal inoculation. We assessed differential expression of pilin using qRT-PCR.

Results: Results. Expression of pilin transcripts measured by qRT-PCR was restored when the phoRP deletion was complemented. The pilin deletion mutant was significantly less able to associate with THP-1-derived macrophages after 2 hours of infection (~2-fold reduction, p value < 0.0001). The pilin deletion mutant was also less able survive within the THP-1-dervived macrophage after extracellular bacteria were killed with antibiotics (~4-fold reduction, p</em> value < 0.0001). CF mice that were infected the pilin mutant had significantly lower number of bacteria within their lungs 7 days after infection compared to mice infected with the parental strain (~6-fold lower, p value < 0.05). Interestingly, there was no reduction seen in non-CF littermate control mice. Also, when competition infections were done where mice (CF or non-CF) were infected with a mix of pilin mutants and parental bacteria, there was no difference in the fitness of the pilin mutant suggesting in vivo complementation of the pilin deletion by a secreted factor.

Conclusions: Conclusions. The phoRP system is required for full virulence of members of the M. abscessus complex. PhoRP regulates the expression of a pilin in a clinical CF M. abscessus isolate, and this pilin is a required for association and survival within macrophages and CF mice

Acknowledgements: This work was supported by Cystic Fibrosis Foundation (SCHAEF2010).

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