Research

Perforin-2 and Innate Immunity

Perforin-2, the product of the Mpeg1 gene, is a major focus of research within the Munson laboratory. Beginning with the foundational work of Dr. Eckhard Podack, our former Chair and colleague, studies at the University of Miami have shown that Perforin-2 is an effector of the innate immune system that limits the spread and proliferation of bacterial pathogens. Its amino-terminus contains a Membrane Attack Complex Perforin (MACPF) domain which is also present in several complement proteins and Perforin. Gram-negative bacteria are killed when complement proteins C6-C9 form pores in the bacterial envelope. Cytotoxic T lymphocytes (CTL) and natural killer cells (NK) use Perforin to form lytic pores in the membranes of tumor and virally infected mammalian cells. Our 2020 publication with collaborator Prof. Robert Gilbert demonstrated that Perforin-2 also forms pores. In a separate study we showed that Perforin-2 is required to breach the envelope of phagocytosed bacteria. In contrast to complement C6-C9 whose activity is restricted to gram-negative bacteria, the bactericidal activity of Perforin-2 is broad spectrum; efficacious against gram-negative, gram-positive, and acid-fast bacteria. Moreover, most mammalian cells have the capacity to express Perforin-2 and this ability endows both immune and non-immune, tissue forming cells with a frontline defense against pathogenic bacteria. Not surprisingly, Perforin-2 deficient mice are severely immunocompromised and rapidly succumb to infectious diseases. We are also working with clinicians who have discovered patients with deleterious Mpeg1 mutations that correlate with increased susceptibility to infectious diseases. To better understand the pivotal role of Perforin-2 within the innate immune system we are using multidisciplinary approaches to elucidate its activation, intracellular trafficking, mechanisms of pore-formation and killing, and crosstalk with other parts of the immune system.

Anti-Perforin-2 Bacterial Effectors

The gene encoding Perforin-2, Mpeg1, has existed for hundreds of millions of years and is present in nearly all animal species; including those within the phylum Porifera (sponges) which evolved 500 million years ago. Thus, Perforin-2 has been protecting even the simplest of animal species from infectious diseases since before dinosaurs walked the planet. The evolutionary longevity of Perforin-2 is a testament to its pivotal role within cell-mediated immunity. However, this has also allowed pathogens ample time to evolve mechanisms that inhibit and subvert Perforin-2’s bactericidal activity.

In theory bacterial pathogens have likely evolved numerous strategies to survive Perforin-2. Some may suppress the expression of Perforin-2. Others may escape the early endo-phagosome before Perforin-2 is fully deployed. Some may prevent phagosome acidification to inhibit the acid-dependent pre-pore to pore transition. Still others may deploy effectors to prevent the intracellular trafficking of Perforin-2 or its maturation. Hypothetically, any step along the Perforin-2 pathway –from PAMP signaling and initial expression to the final step of pore formation– is a potential target for pathogen counter defense. In this respect anti-Perforin-2 effectors can be thought of as molecular probes that help map the Perforin-2 dependent killing pathway through elucidation of an effector’s mechanism of action. Thus, the discovery of anti-Perforin-2 effectors complements other Perforin-2 research within the Munson lab.

Anti-Perforin-2 Nanobodies

As students of immunology most of us were –and many still are– taught that antibody paratopes (antigen binding sites) are formed by the synergistic participation of a heavy and light chain. But in the late 1980s a group of biology students at the University of Brussels discovered a subset of immunoglobulins within camel serum that did not correlate with any known class of antibodies. Subsequent investigations by Hamers-Casterman et al. (Nature 1993) revealed that this novel class of antibodies were devoid of light chains and are present in all camelids; including llamas and alpacas.

In this astonishing class of antibodies the paratope resides solely within the variable region of an individual heavy chain.

Today heavy chain only antibodies are referred to as VHHs, sdAbs, llama-bodies, or nanobodies. VHH -an acronym for Variable Heavy domain of Heavy chain- is the most technically precise term. sdAb is an acronym for Single Domain Antibodies. Llama-bodies is in reference to llamas as the preferred animal for raising VHHs against inoculated antigens. Nanobodies refers to the fact that the constant regions are dispensable since there is no need to pair a heavy chain with a light chain. Thus, the smallest functional unit of this new class of antibodies is ~12 kDa; less than one tenth the size of conventional immunoglobulin. Moreover, once the sequence of a VHH is obtained the antibody can be produced in bacteria such as E. coli; eliminating the effort and expense required for the production of mAbs by culture of mammalian hybridomas. The ability to produce VHHs in bacteria has most recently given rise to rationally designed, fully synthetic libraries of VHHs. Such synthetic libraries can be panned for VHHs with specificity to any given antigen; completely eliminating research animals (llamas) from the production protocol.

Currently the Munson lab is producing synthetic VHHs against human and murine Perforin-2. We are producing VHHs in E. coli and are in the process of validating them in various immunological assays; i.e., FLOW, IF, pull-downs, etc. In addition to our own research needs, our long term goal is to provide validated VHHs to the research community. We are particularly interested in VHHs that can distinguish between three different conformation: (1) monomeric Perforin-2, (2) oligomeric Perforin-2 pre-pores and (3) oligomeric Perforin-2 pores.

Enterotoxigenic E. coli (ETEC)

Approximately 1.7 million people, primarily infants and children, perish from diarrheal diseases every year. For citizens of low–income countries diarrheal diseases are among the ten leading causes of death. Although many viral and bacterial pathogens cause diarrhea, ETEC is prevalent in low–income nations where it is estimated to kill between 300,000 to 700,000 children and infants each year. Another 280 million people are sickened by ETEC annually including travelers from high income countries. Despite decades of research there are no FDA approved vaccines to prevent ETEC infections. Although some vaccines are currently under development and evaluation, at least one initially promising vaccine has failed in a large scale clinical trial. Genome plasticity and strain heterogeneity is a further obstacle to vaccine development. Thus, the prospects for ETEC vaccines are uncertain. To provide potential alternative approaches to limit the morbidity and mortality associated with diarrheagenic pathogens the Munson lab is characterizing ETEC virulence factors and their regulation.

Canonical Type 1 Secretion Systems (T1SS) of gram-negative bacteria accept client proteins from the cytoplasm and transport them across the inner membrane, through the periplasm, and finally across the outer membrane in a single translocation step. The prototypical T1SS is the hemolysin system of E. coli that exports hemolytic toxin (Thomas, Holland and Schmitt 2014). All five Cex proteins, CexPABCD, are required for the secretion of CexE to the outer membrane and some are homologous to components of T1SS. Most notable is CexA which is a member of the TolC family. As has been shown for TolC, CexA likely forms a trimeric beta-barrel through the outer membrane. T1SS also have ATP binding cassettes (ABCs) and hydrolyze ATP to drive protein secretion. In the Cex system the ABC resides within CexC and it is the putative ATPase of the secretion system.

However the Cex secretion system is a noncanonical T1SS because it accepts its client protein -CexE- from the periplasm, not the cytoplasm. We have shown that CexE has an amino-terminal signal peptide (Pilonieta, Bodero and Munson, 2007) and is transported across the inner membrane by the SecYEG translocase. As it enters the periplasm its signal peptide is removed by a signal peptidase. What happens next to CexE is not yet clear. The protein may undergo further maturation; in particular the formation of an internal disulfide bond and additional post-translational modifications. Eventually the Cex secretion accepts periplasmic CexE for final transport across the outer membrane. We have chosen to designate this system as a Type1p Secretion System (T1pSS) because the Cex secretion system is noncanonical. Here “p” denotes that this system accepts a periplasmic client.

Current Questions

• How does the Cex secretion system recognize its client?
• Do the periplasmic domains of CexD or CexP bind CexE or other secretion components?
• Is post-translational modification of CexE required for secretion?
• How is the hydrolysis of ATP by cytoplasmic CexC coupled to the secretion of CexE?
• Is secretion regulated by specific external or internal stimuli?
• What is the stoichiometry of the assembled secretion system?
• Does the secretion system assemble in the absence of CexE?
• Does CexE change the stoichiometry of the secretion system or its intermolecular associations?

The expression of CexE and its secretion system is dependent upon the transcription factor Rns. This transcription factor also regulates the expression of adhesive pili and other virulence factors. Thus, Rns is the master regulator of ETEC virulence. A recent collaboration with the Kull lab at Dartmouth has also revealed that Rns may be a therapeutic target to limit ETEC pathogenesis. Our collaboration has shown that medium chain fatty acids are potent inhibitors of Rns and thus abolish the expression of CexE, its secretion system, adhesive pili, and other Rns-dependent virulence factors (Midgett et al., Nature Scientific Reports 2021). Currently we are developing animal models to evaluate medium chain fatty acids as prophylactics against bacterial diarrheal diseases. We are also evaluating the effects of fatty acids on the virulence regulons of other enteric pathogens and conducting genetic, molecular and structural studies of their regulators.