Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication is followed by macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here in Chapter 2, we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the closely related extracellular fungal pathogen Blastomyces dermatitidis. We determine the de novo structures of Cbp1 from Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold, consisting of a helical dimer arrangement where two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type E. africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a primary intracellular human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor. Taken together, these data imply that Cbp1 is a rapidly evolving fungal virulence factor that localizes to the cytosol to trigger host cell lysis. We also show that Cbp1 is not the only virulence factor that enters the macrophage cytosol. In Chapter 3, we identify other virulence factors including a Cerato-platanin domain containing protein (Hc Asp F13) and Yps-3, are also found in the macrophage cytosol and contribute to optimal lysis In Chapter 4, we identify a family of putative cysteine-knot containing proteins that are specifically expanded in Hc species using a new, unbiased algorithm known as KNOTTIN Finder. One putative cysteine-knot, Rockstar10, contributes to optimal lysis of macrophages during infection. Taken together, these data demonstrate that Hc relies on a strategy of secreting cysteine-rich proteins into the macrophage cytosol in order to potentiate the lysis of its host cell to further dissemination.

Bacteria deploy multiple defenses to prevent mobile genetic element (MGEs) invasion. CRISPR-Cas immune systems feature RNA-guided nucleases that target MGEs, which counter with anti-CRISPR (Acr) proteins. Our understanding of the biology and co-evolutionary dynamics of the common Type I-C CRISPR-Cas subtype has lagged because it lacks an in vivo phage-host model system. Here, we show the anti-phage function of a Pseudomonas aeruginosa Type I-C CRISPR-Cas system encoded on an active conjugative pKLC102 island, and the inhibition of this system by multiple distinct MGEs encoding a diverse repertoire of Type I-C Acr proteins. Seven distinct AcrIC proteins were identified, with four of them, including previously described DNA mimic AcrIF2 (now AcrIF2*), surprisingly also inhibiting other P. aeruginosa CRISPR-Cas subtypes (Type I-E or I-F). Dual inhibition comes at a cost, however, as the simultaneous expression of Type I-F and Type I-C systems rendered phages expressing AcrIF2* more sensitive to targeting. This effect was exacerbated by mutagenesis of AcrIF2’s acidic residues, which made AcrIF2 defective for Type I-C inhibition, but only when in competition with the Type I-F complex. Like AcrIF2*, five of the AcrIC proteins block DNA binding by the crRNA-guided Cascade complex, while two function downstream of DNA binding, likely preventing Cas3 recruitment or activity. One such inhibitor, AcrIC3, is found encoded alongside bona fide Cas3 inhibitors, AcrIF3 and AcrIE1 in conjugative elements, forming an “anti-Cas3” cluster. Collectively, our findings demonstrate an active battle between an MGE- encoded CRISPR-Cas system and its diverse MGE targets. On the technological angle, CRISPR–Cas enzymes have enabled programmable gene editing in eukaryotes and prokaryotes. However, the leading Cas9 and Cas12a enzymes are limited in their ability to make large deletions. Here, we used the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering in bacteria. DNA cleavage guided by a single CRISPR RNA generated large deletions (7–424 kilobases) in Pseudomonas aeruginosa with near-100 percent efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837 kb (13.5 percent). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade–Cas3, but not Cas9. A transferable ‘all-in-one’ vector was functional in Escherichia coli, Pseudomonas syringae and Klebsiella pneumoniae, and endogenous CRISPR–Cas use was enhanced with an ‘anti-anti-CRISPR’ strategy. P. aeruginosa Type I-C Cascade–Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.

To survive the harsh conditions of their environment, microbes have evolved protection mechanisms that counteract the most lethal of threats: damage to essential biosynthetic machinery and infiltration by foreign invaders. Surveillance mechanisms monitor fundamental biosynthetic processes—including DNA replication, mRNA transcription, and protein translation—to ensure their accuracy and limit the accumulation of incorrectly synthesized macromolecules in the cell. Immunity mechanisms, on the other hand, prevent cellular infiltration by external invaders such as viruses. In the first half of this study, I investigated a surveillance pathway that detects ribosomes stalled during protein translation and marks the incomplete nascent proteins for degradation. In yeast, this pathway was known to involve Carboxy-terminal Alanine and Threonine (CAT) tailing: a non-canonical, mRNA-independent peptide synthesis mechanism. However, the mechanism of CAT tailing and its role in maintaining protein homeostasis was unknown. Here, I show that CAT tailing requires the catalytic activity of fragmented 60S ribosomes and charged tRNAs, but is otherwise distinct from canonical protein synthesis. Moreover, CAT tailing of the incomplete nascent protein exposes a lysine residue that is modified with ubiquitin, a tag for protein destruction. In the second half of this study, I investigated the bacterial CRISPR-Cas9 immune system in Listeria monocytogenes, which detects and destroys invading phage (virus) DNA. The phage protein, AcrIIA1, was known to inactivate CRISPR-Cas9 but its mechanism was uncharacterized. Here, I show that AcrIIA1 binds to Cas9 with high affinity via the catalytic HNH domain to induce Cas9 degradation, thereby protecting the Listeria genome during lysogeny but not during lytic growth. AcrIIA1 also directly represses transcription of the anti-CRISPR locus and is therefore required for optimal phage replication. Finally, bacterial hosts have co-opted AcrIIA1 homologs that potentially act as “anti-anti-CRISPRs” by blocking the deployment of phage anti-CRISPRs.

There are approximately 5 x 1030 bacteria on Earth [1]. These microorganisms are found in every type of environment, soil, aquatic, air, on every surface in our world. In fact, there is roughly 1 million times more bacteria on earth than stars in the observable universe [2]. These microbes represent a universe on a microscopic scale. Life as a bacterium is challenging and dangerous. Small viruses capable of infecting these bacteria (called bacteriophages or phages), can turn a happy and healthy bacterium to a sick and dying one in less than 30 minutes. Before long, the virus has multiplied in number and an outbreak poses a great risk to the bacterial population. However, some bacteria are immune or protected from certain phages because they encode anti-phage genes or defense genes. This phage-bacteria warfare has been undergoing for millions of years at a scale larger than the observable universe. This has led the evolution of various defense systems and mechanisms that protect bacteria from phages, and conversely the evolution of diverse anti-defense genes that phages use to counteract the defenses. This arms race can be traced by analyzing the genomes of bacteria and phages. In 2011, it was observed that these defense systems are encoded near each other [3]. This is a remarkable observation given the null hypothesis, which is the placement of genes along the genome is random. This led to the term “defense islands” which are genomic regions that encode genes related to anti-phage immunity. One of the most common systems found in these defense islands are restriction modification (RM) systems which generally consists of two components, a restriction enzyme and a modification enzyme (methyltransferase). The RM system recognizes small patterns of DNA and modifies it. This allows the bacteria to mark its own DNA and degrade any DNA without the modification. This allows for protection against foreign DNA injected during a phage infection [4]. This system is by far the most common system seen amongst bacteria. Another remarkable example of an immune system is the CRISPR system which represents an adaptative immune system. CRISPR uses RNA molecules and Cas proteins to recognize specific patterns of DNA from the invading phage. Fragments from the invading phage can be integrated into the bacterium’s genome in the CRISPR array [5]. These two systems alone have been the focus of research for years and has led to the development of several biomedical tools. Discovering more of these systems can help us understand the phage-bacteria arms race and has potential to uncover new biology and development of new technologies.Defense Islands are a focus for discovering new immune systems. Instead of testing the several thousand genes of a bacteria for anti-phage function, defense islands can narrow the search space to the several genes within the island. This has led to the discovery of several immune since 2018 [6]. Defense Islands have been considered potentially mobile elements that are horizontally transferred between bacteria, something that has been known to exist for antibiotic resistance genes. Additionally, defense islands have been considered to be randomly placed in a genome. However, closely related bacterial isolates are known to carry completely different immune systems despite most their genome being identical. These defense islands may utilize the same genomic site for closely related isolates. Indeed, this was observed in the work outlined in the first chapter of this dissertation. This allows for better predications of immune systems because instead of predicting the system itself, it predicts the genomic locus that is likely to encode an immune system, agnostic to previously known systems, which other methods rely on. In Pseudomonas aeruginosa, there are two predominate defense hotspots, (core defense hotspot 1 and 2, cDHS1/2) which is a core and conserved region found in every P. aeruginosa isolate that is hotspot for several defense islands. These hotpots are likely built by the accumulation of several mobile defense islands or systems in this region. Phages also have the ability to evolve proteins that are antagonistic to bacteria. Anti-defense genes like AcrIF11, an anti-CRISPR that prevents phages from being targeted from the CRISPR Cas Type I-F system. ArdA, an anti-RM protein that inhibits RM systems and allows phages to infect bacteria despite having an RM system. A recent study reported seven novel anti-CRISPR protiens that protect phage from the type IV-A system, But, when we looked at the study, we found several issues. We tested the Acr sequences in E. coli and mammalian cells but did not find any anti-Cas13a activity. This makes us question the original study’s claim. The details on findings is the topic of chapter 2 of this dissertation.

The human gut microbiota encompasses all the microorganisms that reside in the gastrointestinal tract. Bacteria, its most abundant member, are known to have an impact on their human host. Bacteriophages (phages), also abundant within the microbiota, can potentially alter gut composition yet their interactions with bacteria remain poorly understood, particularly with respect to CRISPR-Cas immunity. Here, we focus on gut bacterium Eggerthella lenta: attempting to develop genetic tools, investigating its CRISPR-Cas system, and isolating phages against it. While developing a transformation protocol, we discovered endogenous plasmids and antibiotic resistance genes that we used for constructing shuttle vectors. We demonstrated that its CRISPR-Cas system is transcribed and sufficient for targeting of DNA. Furthermore, we assembled a database to identify protospacers that revealed matches for a majority of spacers and uncovered “hyper-targeted” phage and determined the mismatch tolerance of observed spacer/protospacer pairs. To study phage interactions with microbiome members and gut bacterial evasion of phages, we screened wastewater for phage against E. lenta. We isolated, sequenced, and annotated 4 novel and highly related phages. Using the phage to target a sensitive strain in gnotobiotic mice didn’t have an impact on bacterial abundance, despite their ability to lyse E. lenta in vitro. Our work lays a foundation for the development of tools for E. lenta, an understanding of its CRISPR-Cas system and characterization of novel phages.

Bacteria and the viruses that infect them (bacteriophages/phages) are locked in an ancient evolutionary arms race. Bacteria use CRISPR-Cas systems as a form of anti-phage immunity and phages encode “anti-CRISPR” proteins that antagonize CRISPR-Cas function. Anti-CRISPRs are high-affinity CRISPR-Cas inhibitors, but face a great challenge in rapidly neutralizing all the CRISPR-Cas complexes in the cell upon phage infection. I show that CRISPR defeats phages with anti-CRISPRs in >90% of infection events, and rare successes occur only when multiple phages cooperate to infect the same cell. Failed infections “immunosuppress” the bacterium by the production of anti-CRISPRs prior to phage destruction, increasing chances of survival for other co-infecting phages. This is the first description of altruism in viruses. This cooperative strategy for CRISPR-Cas neutralization is completely dependent on phage multiplicity of infection: if the concentration of phage is too low for coinfections to occur at an appreciable rate, the phage population will go extinct. While this strategy may be suitable for some classes of mobile elements, others may be unable to obtain cooperation thresholds, necessitating enzymatic or hyper-potent inhibitors. In searching for new mechanisms of CRISPR-Cas neutralization, we have now discovered anti-CRISPRs that function substoichiometrically, one of which has been shown to be a multi-turnover enzyme. Furthermore, I show that some phages have hijacked a transcriptional repressor of CRISPR-Cas immunity, and deploy it to limit CRIPSR-Cas biogenesis. The CRISPR/anti-CRIPSR arms race is a hotbed for evolutionary innovation, and is a continuous source of novel and inventive mechanisms of immune neutralization.