Research

Discovering the molecular mechanisms underlying how bacteria sense their environment

Tiffany joined Dr. Anupama Khare’s systems biology lab to expand her research to more complex multispecies systems which accurately reflect the lifestyle of many bacteria. Sensing of other microbes and behavioral responses are prevalent in microbial communities. The objective of her postdoc research is to elucidate polymicrobial interactions among co-infecting cystic fibrosis (CF) bacterial pathogens. She developed a strategy to systematically identify molecules from one species that are sensed by another and applied these approaches to two often co-isolated CF pathogens: Pseudomonas aeruginosa and Staphylococcus aureus. She found that P. aeruginosa can sense multiple staphylococcal secreted products. Additionally, she defined the S. aureus genes that are responsible for the production of the sensed molecules, and the pathways in P. aeruginosa that are induced upon sensing each molecule. She discovered specific molecular mechanisms underlying beneficial and antagonistic interspecies interactions, providing a better understanding of cooperation and competition between these two co-infecting pathogens. Her current efforts are focused on the specific molecular mechanisms that regulate behavioral responses in interspecies interactions.

Zarrella & Khare. 2022 PLOS Biology Cover Article.

Postdoc Profile in I Am Intramural blog

Research featured on CellOut Podcast. Episode: Jargon Strikes Back. 2022.

For her graduate studies, Tiffany joined the lab of Dr. Guangchun Bai at Albany Medical College to decipher the role of cyclic di-adenosine monophosphate (c-di-AMP) in Streptococcus pneumoniae. c-di-AMP was newly discovered bacterial second messenger involved in pathogenesis at the time of this work. S. pneumoniae infections are challenging to prevent and treat due to its ability to withstand many stress conditions, to cause infections in different organs, to undergo capsule switching which allows vaccine evasion, and to acquire antibiotic resistance genes. Tiffany led two projects to expand understanding of c-di-AMP signaling in S. pneumoniae. Tiffany helped characterize the role of CabP, a Trk (transporter of K⁺) family protein, and its cognate transmembrane transporter, TrkH, in potassium uptake. In addition, Tiffany discovered that the c-di-AMP binding protein CabP affects c-di-AMP homeostasis. This is the first report of a c-di-AMP effector protein modulating c-di-AMP levels. In the second project, Tiffany found that c-di-AMP modulates genetic competence, a program that affects DNA uptake. This finding is critical to understanding how to subvert antibiotic resistance and capsule switching. In addition, Tiffany characterized the role of c-di-AMP in stress survival, which is different than what has been described in other pathogens and suggests a divergence of c-di-AMP signaling targets in S. pneumoniae. Collectively, her work provides insights into the molecular mechanisms by which c-di-AMP affects the pneumococcal stress response and competence.

Book chapter: Zarrella & Bai. 2020. in Microbial cyclic di-nucleotide signaling

Minireview: Zarrella & Bai. 2020. Journal of Bacteriology.

Research articles:

Bai et al. 2014. Journal of Bacteriology.

Zarrella et al. 2018. Journal of Bacteriology.*

Zarrella et al. 2020. Journal of Bacteriology.

*Commentary about Tiffany's work

As a research technician, Tiffany worked in the lab of Dr. Karsten Hazlett at Albany Medical College. There she studied host adaptation and virulence regulators in Francisella tularensis. F. tularensis is a bacterial pathogen that can cause disease with fewer than 25 colony-forming units, is readily aerosolized, and thus has been proposed as a potential biowarfare agent. This pathogen naturally survives in many different hosts, including mammals, insects, arthropods, and protozoans, for which the environmental conditions vary and Francisella must adapt. However, studies performed with in vitro culture find discordant results from mammalian infection models. The aim of this work was to ascertain surface protein accessibility in in vitro conditions that mimic mammalian-adapted F. tularensis. Utilizing an immuno-pulldown assay, by applying antibodies to survey the surface-exposed proteins on bacteria, they established that host-adapted F. tularensis produces more surface carbohydrates than bacteria grown in medium containing branched chain amino acids, thereby shielding outer membrane proteins from being recognized by the immune receptor TLR2 and complement. These results demonstrated the stealth of Francisella when grown in the mammalian host, which is not shown in non-host-adapted bacteria. To globally analyze differential protein expression of F. tularensis in these growth conditions, Tiffany prepared samples for proteomics analysis (performed by Dr. Robert Ernst’s group) which revealed an overwhelming increase in proteins expressed from the Francisella pathogenicity island and in lipopolysaccharide-producing enzymes. This work 1) defines an interplay between F. tularensis and immune surveillance, 2) describes the importance of using the appropriate in vitro growth conditions for research, and 3) provides a significant contribution for future therapeutic studies of the pathogen.

In addition, Tiffany also worked with Dr. Hazlett, Dr. Timothy Sellati, and Dr. Edmund Gosselin to develop a vaccine platform and patent. Vaccine production for biothreats can be time-consuming and expensive due to testing specific antigens and supplying adjuvants which may affect toxicity. There is a need for a universal vaccine design that can be applied to bacteria to direct antigens to immune cells for processing and presentation. This vaccine platform is devised to express a lipoprotein that serves as an anchor to deposit surface accessible complement on Gram-negative bacteria in order to target the whole cell with species-specific immunogens to antigen-presenting immune cells. Specifically, Francisella tularensis lipoprotein Tul4 (a TLR2 agonist) was fused to multiple copies of the complement product C3d and expressed in Gram-negative pathogens. Tiffany helped design the vector and developed some of the preliminary results for the vaccine platform patent application. After constructing the vaccine candidate strains, Tiffany observed successful accessibility of the immunogenic proteins on the bacterial cell surfaces of Escherichia coli, F. tularensis, and Yersinia pestis. This design is advantageous because it is adjuvant-free and can be applied for use in many different bacterial pathogens.

Zarrella et al. 2011. PLOS One.

Holland et al. 2017. Frontiers in Microbiology.

Patent