Jochen Zimmer - US grants

DESCRIPTION (provided by applicant): Bacterial biofilms are best described as multi-cellular, usually sessile, bacterial aggregates stabilized by extracellular polysaccharides and are implicated in a number of pathogenic conditions. For example, chronic biofilm infections of Pseudomonas aeruginosa are commonly found in cystic fibrosis patients. Endocarditis, the inflammation of the heart chamber and valve, can be caused by biofilms of Staphylococcus aureus and Streptococcus viridans, and dental plaques are biofilms of Streptococcus mutans and sanguinis on the surface of the teeth. Biofilm bacteria pose a particular risk to human health because of their low susceptibility to common antibiotics and the host immune system. The polysaccharides typically found in bacterial biofilms include ¿-1,6 linked N-acetyl-glucosamine, alginate, and cellulose. By using the bacterial cellulose synthase machinery as a model system, we propose to determine how extracellular polysaccharides are synthesized and transported across the bacterial cell envelope. This process is particularly interesting because extracellular polysaccharides are synthesized inside the cell from nucleotide diphosphate-activated precursors and can grow to several microns in length, yet they are efficiently secreted to reach the site of their biological function. We combine the tools of molecular and structural biology to first, identify the essential components required for cellulose synthesis and membrane translocation, second, to reconstitute cellulose biosynthesis in vitro from purified components, and third, to determine the 3-dimensional structure of the catalytically active subunit of the cellulose synthase complex. We developed a novel in vitro asay for celulose synthesis, demonstrating that the iner membrane components of the cellulose synthase machinery (BcsA and BcsB) are required for celulose synthesis and translocation. While BcsA is the catalytically active subunit, BcsB is an auxiliary subunit that most likely associates with BcsA; however, its precise role during cellulose synthesis is unclear. Therefore, based on our in vitro assay, we propose to define the minimal core of the BcsB subunit required for cellulose synthesis (Aim 1). To ultimately prove that the BcsA and BcsB components are sufficient for cellulose synthesis and translocation, we have to reconstitute the reactions in vitro from purified components. Thus, our second aim is to purify the BcsA and BcsB subunits and to reconstitute cellulose synthesis and membrane translocation in proteoliposomes. To gain mechanistic insights into the process of cellulose biosynthesis, biochemical data obtained from aims 1 and 2 must be integrated with structural information on the key enzymes. Therefore, the third aim of this proposal is to solve the 3-dimensional structure of the cellulose synthase subunit BcsA by x- ray crystallography. Overall, we undertake a multi-disciplinary approach to reveal how one of nature's most abundant polymers is synthesized and translocated across biological membranes.

DESCRIPTION (provided by applicant): The extracellular matrix in vertebrates provides structural support to the cell, aids in osmo-regulation, and is particularly important in mediating cell-cell interactions in soft connective tissues, such as cartilage and skin. A major component of the extracellular matrix is hyaluronan (HA), which is an extracellular linear polysaccharide containing alternating N-acetylglucosamine (NAG) and glucuronic acid (GA) residues. HA affects many physiological processes, from cell adhesion and migration to cell differentiation and embryological development. Because of its broad impact on human physiology, a large number of pathological conditions, including many forms of cancer, autoimmune diseases, inflammatory processes, and rheumatoid arthritis, correlate with altered expression levels of HA. On a molecular level, HA is produced inside the cell by the membrane-embedded hyaluronan synthase (HAS). HAS is a remarkable enzyme. It not only catalyzes the synthesis of HA from UDP-activated substrates, but it also transports the growing polymer across the cell membrane to deposit it within the extracellular matrix. In order to accomplish this task, HAS has to fulfill several functions. The enzyme binds the substrates UDP-NAG and -GA, it catalyzes the glycosyl transfer reaction to form HA, and it translocates the growing polymer across the cell membrane through a pore formed by its own transmembrane region. To understand how HA exerts its physiological function and to produce HA polymers with defined properties for biomedical applications, we must first unravel how HAS synthesizes HA and how it deposits the polymer in the extracellular matrix. To this end, we propose three aims that will reveal the assembly of biologically active HAS subunits in native lipid membranes, will identify the interactions between HAS and the translocating HA polymer, and will allow us to determine the structure of HAS by X-ray crystallography. First, we will combine co-immunoprecipitation studies with chemical cross-linking and photobleaching techniques to visualize HAS oligomers in native membranes. The low-resolution structural data will then be integrated with high-resolution structures of monomeric HAS to reconstruct the native, membrane-embedded HAS oligomer. Second, the interactions of HAS with the translocating HA polymer will be mapped by introducing UV-inducible cross-linkers into the TM-region of HAS. Cross-linking during HA translocation will identify positions that are in close proximity to the polysaccharide, thus delineating the physico-chemical properties of the HA transmembrane channel. Third, biochemical and low resolution structural data will be integrated with a high-resolution structure of HAS obtained by X-ray crystallography. Determining the structure of HAS both in a detergent-solubilized but also in a membrane-embedded state will reveal the architecture and oligomeric form of the synthase, allowing us to delineate the mechanism by which this marvelous enzyme synthesizes one of the most abundant extracellular polysaccharides in the human body.

DESCRIPTION (provided by applicant): The extracellular matrix in vertebrates provides structural support to the cell, aids in osmo-regulation, and is particularly important in mediating cell-cell interactions in soft connective tissues, such as cartilage and skin. A major component of the extracellular matrix is hyaluronan (HA), which is an extracellular linear polysaccharide containing alternating N-acetylglucosamine (NAG) and glucuronic acid (GA) residues. HA affects many physiological processes, from cell adhesion and migration to cell differentiation and embryological development. Because of its broad impact on human physiology, a large number of pathological conditions, including many forms of cancer, autoimmune diseases, inflammatory processes, and rheumatoid arthritis, correlate with altered expression levels of HA. On a molecular level, HA is produced inside the cell by the membrane-embedded hyaluronan synthase (HAS). HAS is a remarkable enzyme. It not only catalyzes the synthesis of HA from UDP-activated substrates, but it also transports the growing polymer across the cell membrane to deposit it within the extracellular matrix. In order to accomplish this task, HAS has to fulfill several functions. The enzyme binds the substrates UDP-NAG and -GA, it catalyzes the glycosyl transfer reaction to form HA, and it translocates the growing polymer across the cell membrane through a pore formed by its own transmembrane region. To understand how HA exerts its physiological function and to produce HA polymers with defined properties for biomedical applications, we must first unravel how HAS synthesizes HA and how it deposits the polymer in the extracellular matrix. To this end, we propose three aims that will reveal the assembly of biologically active HAS subunits in native lipid membranes, will identify the interactions between HAS and the translocating HA polymer, and will allow us to determine the structure of HAS by X-ray crystallography. First, we will combine co-immunoprecipitation studies with chemical cross-linking and photobleaching techniques to visualize HAS oligomers in native membranes. The low-resolution structural data will then be integrated with high-resolution structures of monomeric HAS to reconstruct the native, membrane-embedded HAS oligomer. Second, the interactions of HAS with the translocating HA polymer will be mapped by introducing UV-inducible cross-linkers into the TM-region of HAS. Cross-linking during HA translocation will identify positions that are in close proximity to the polysaccharide, thus delineating the physico-chemical properties of the HA transmembrane channel. Third, biochemical and low resolution structural data will be integrated with a high-resolution structure of HAS obtained by X-ray crystallography. Determining the structure of HAS both in a detergent-solubilized but also in a membrane-embedded state will reveal the architecture and oligomeric form of the synthase, allowing us to delineate the mechanism by which this marvelous enzyme synthesizes one of the most abundant extracellular polysaccharides in the human body.

Many  bacterial  pathogens  secrete  virulence  factors  to  perform  a  variety  of  biological  functions,  including  formation  of  cytolytic  pores,  modulation  of  host  immune  responses,  or  scavenging  nutrients.  The  Type  1  secretion  system  (T1SS)  is  widespread  among  pathogens  and  translocates  toxins  across  the  cell  envelope  in  Gram-­negatives.  One  T1SS  example  is  found  in  hemorrhagic  E.  coli  strains  that  secrete  hemolysin,  a  pore  forming  toxin.  T1SS  consist  of  inner  and  outer  membrane  components.  Integrated  into  and  anchored  to  the  inner membrane is an ABC transporter and a periplasmic membrane fusion protein, respectively, of which the  ABC  transporter  energizes  protein  translocation.  These  components  partner  with  an  outer  membrane  porin  to  facilitate  translocation  across  the  outer  membrane.  T1SS  substrates  can  be  extremely  long,  ranging  in  size  from  40  to  >900kDa.  Despite  their  lengths,  the  polypeptides  are  directly  channeled  from  the  cytosol  to  the  exterior of the cell. We already solved the crystal structure of the T1SS ABC transporter PrtD in a resting state.  Because  PrtD  is  only  functional  in  a  fully  assembled  T1SS  and  to  gain  insights  into  the  mechanism  of  T1SS-­ protein  secretion,  we  propose  to  determine  the  structure  of  a  stalled  T1SS  translocation  intermediate  by  cryo  electron  microscopy  (Aim  1).  Substrate  translocation  will  be  stalled  by  fusing  a  stably  folded  domain  to  the  substrate?s N terminus. T1SS translocate substrates C terminus first, which forms a stable Ca2+ binding domain  in the extracellular milieu. Thus, by introducing a stably folded domain to the substrate?s N terminus, the T1SS  is  trapped  between  folded  cytosolic  and  extracellular  domains.  To  generate  these  T1SS  translocation  intermediates, we assembled a functional T1SS in vivo from heterologously expressed and individually tagged  components.  This  system  secretes  the  substrate  PrtG  into  the  culture  medium  and  allows  trapping  of  translocation intermediates with GFP-­fused substrates.   Another  defense  mechanism  employed  by  many  Gram-­negative  pathogens  is  the  modification  of  lipopolysaccharides (LPS) with complex carbohydrates (O-­antigens), thereby preventing complement-­mediated  killing. O-­antigens are mostly linear polysaccharides about 100 to 400 sugar units long. A common biosynthetic  pathway involves the synthesis of fully assembled and lipid-­anchored O-­antigens on the cytosolic leaflet of the  inner  membrane,  after  which  the  polymer  is  transported  to  the  periplasmic  side  by  a  specific  ABC  transporter  before being attached to the outer core oligosaccharide of LPS. We already determined the crystal structure of  the  O-­antigen-­translocating  ABC  transporter  in  an  open  conformation,  revealing  a  continuous transmembrane  channel suitable to accommodate a polysaccharide chain. To unravel the mechanism by which this polymer is  translocation, we propose to determine the transporter?s substrate-­bound structure using O-­antigens of defined  lengths,  synthesized  in  vivo  or  in  vitro  (Aim  2).  Substrate-­bound  complexes  will  be  generated  by  co-­ crystallization or crystal-­soaking experiments with the purified substrates.                         

Antibiotic-­resistant  pathogens  pose  a  significant  threat  to  human  health.  The  NIH  identified  several  Gram-­ negative  species  of  particular  concern  due  to  increasing  antibiotic  resistances.  The  cell  envelope  of  Gram-­ negative  bacteria  consists  of  two  membranes  and  lipopolysaccharides  (LPS)  form  an  important  component  of  the  extracellular  leaflet  of  the  outer  membrane.  LPS  are  important  cell  wall  components  that  control  diffusion  across the outer membrane, stabilize the cell envelope, and assist in escaping host immune defenses, among  other functions. Impaired LPS biosynthesis correlates with increased susceptibility to antimicrobial treatments.  LPS  contain  a  conserved  core,  consisting  of  lipid-­A  attached  to  an  oligosaccharide  backbone,  and  a  hypervariable  region,  called  the  O  antigen.  O  antigens  are  primarily  linear  complex  carbohydrates  that  reduce  the  efficacy  of  complement-­mediated  cell  lysis  and  phagocytosis  as  part  of  the  innate  immune  response.  O  antigens  are  synthesized  via  two  fundamentally  different  mechanisms.  One  pathway  relies  on  assembling  the  polymers  from  short  oligosaccharides  in  the  periplasm,  the  other  involves  moving  fully-­assembled  O  antigens  from the cytosolic to the periplasmic side of the inner membrane with the help of an ABC transporter. Not only  is  ABC  transporter-­mediated  secretion  of  O  antigens  an  important  process  for  microbial  pathogenicity,  the  transport  of  a  substrate  several  times  the  size  of  the  ABC  transporter  itself  is  fascinating  from  a  molecular  level.  Taking  advantage  of  an  already  determined  O  antigen-­translocating  ABC  transporter  structure,  we  propose  a  structural  biology  approach  to  unravel  the  mechanism  of  O  antigen  translocation  and  to  identify  unique features of the O antigen that regulate transporter activity.    ABC  transporters  use  ATP  binding  and  hydrolysis  to  cycle  between  conformations  that  mediate  substrate  translocation. Our O antigen ABC transporter structure represents a nucleotide-­free conformation, in which the  transporter  forms  a  continuous  channel  across  the  membrane  that  could  accommodate  a  translocating  O  antigen.  We  speculate  that  conformational  changes  associated  with  nucleotide  binding  induce  O  antigen  translocation  by  about  1-­2  sugar  units  per  ATP  hydrolyzed.  To  reveal  the  molecular  mechanism  of  O  antigen  translocation,  we  seek  to  determine  the  structure  of  the  ABC  transporter  in  a  nucleotide-­bound  closed  conformation  (Aim  1).  Further,  many  bacterial  species  signal  completion  of  O  antigen  biosynthesis  by  modifying  the  polymer?s  growing  end  with  specific  groups,  such  as  carbohydrate,  phosphate,  or  methyl  moieties.  These  ?capped  O  antigens?  can  only  be  exported  by  transporters  containing  a  carbohydrate-­binding  domain  (CBD)  attached  to  their  nucleotide-­binding  domain.  This  domain  was  removed  from  our  O  antigen  transporter  to  facilitate  crystallization.  To  reveal  how  the  CBD  binds  its  substrate  and  modulates  transporter  functions, we propose to determine the structure and substrate binding properties of the isolated CBD (Aim 2A)  and to determine the architecture of the CBD-­containing full-­length O antigen transporter (Aim 2B).        

Essentially all living systems produce cell surface structures to rigidify cells, form protective coats, or facilitate cell adhesion and migration. Microbial ?cell walls? usually perform protective functions for survival under detrimental conditions, to reduce the efficacy of their host?s innate immune response, or to form 3-dimensional meshworks, called biofilms. Common building materials for these extracellular structures are polysaccharides that either function on their own or are integrated with other polymers into elaborate composite materials. Mucoid Group A Streptococci produce a thick polysaccharide capsule that consists of hyaluronan (HA). HA is an acidic hetero-polysaccharide primarily produced by vertebrates as an abundant component of the extracellular matrix in soft connective tissues, cartilage, and the vitreous of the eye. Because HA is non- immunogenic, microbial HA capsules are an efficient mechanism to escape complement mediated killing, thereby contributing significantly to streptococcal virulence. Group A streptococcal infections can cause severe illnesses, including rheumatic fever and necrotizing fasciitis. We seek to determine the mechanism by which streptococcal HA capsules are formed. HA is synthesized by a membrane-embedded enzyme (HAS) that performs two tasks. It functions as a (1) glycosyltransferase to synthesize HA from UDP-activated substrates and (2) translocase that secretes HA across the membrane through a channel formed by its own membrane-spanning region. How HAS couples these reactions to secrete an acidic polymer up to ~100,000 sugar units long is currently unknown. The proposed research takes advantage of our detailed biochemical analyses of streptococcal HAS. We demonstrated that the enzyme functions as an obligate dimer in which two protomers form a single HA polymer and likely also a HA channel at their interface. This enzyme complex can be purified and reconstituted into planar membrane bilayers, called nanodiscs, which are excellent membrane surrogates for biochemical and structural analyses. We propose to develop a toolset that will allow us to determine the HAS structure at different states during HA biosynthesis. To this end, under Aim 1 we will generate conformation sensitive Fab antibody fragments that specifically recognize 3-dimensional epitopes of HAS. A primary focus will be on identifying Fab fragments that interact with a single HAS copy in the context of a dimeric assembly, which is expected to facilitate structural analyses by cryo electron microscopy. Further, Fab binders will be selected that recognize and stabilize the HAS dimer interface, which are expected to aid in protein crystallization. In Aim 2, we will generate HAS hyaluronan translocation intermediates to (1) identify the polysaccharide length spanning the enzyme?s transmembrane channel, (2) monitor polymer release from the synthase, and (3) allow structure determination by cryo electron microscopy. Combined, our research will provide a complete toolset necessary to obtain structural snapshots of bacterial hyaluronan biosynthesis along its catalytic cycle.

Essentially all living systems produce cell surface structures to rigidify cells, form protective coats, or facilitate cell adhesion and migration. Microbial ?cell walls? usually perform protective functions for survival under detrimental conditions, to reduce the efficacy of their host?s innate immune response, or to form 3-dimensional meshworks, called biofilms. Common building materials for these extracellular structures are polysaccharides that either function on their own or are integrated with other polymers into elaborate composite materials. Capsular polysaccharides (CPS) are abundant among Gram-negative and ?positive bacteria. The polymers form dense extracellular structures that limit diffusion, aid in osmoregulation, and form thick protective coats around the cell. Some CPS mimic host glycans, thereby disguising potent pathogens under an immunologically invisible coat. The polymers are synthesized and deposited on the cell surface by two fundamentally different pathways. One assembles the polymer in the periplasm from short lipid-linked precursors and translocates it across the outer membrane (OM) concomitantly. In the ABC transporter- dependent pathway, however, the CPS is synthesized intracellularly on a lipid anchor and transported after its completion through a secretion system that spans the inner and the OM. The molecular and mechanistic mechanisms of both pathways remain poorly understood. To aid the development of novel antibiotic strategies, we seek to establish a detail structure-function analysis of the abundant ABC transporter-dependent CPS biosynthesis pathway. Our approach is two-pronged. First, we seek to establish a robust genetically tractable model system for CPS secretion (Aim 1A and B). Second, we will complement our functional analyses with detailed structural insights into the CPS ABC transporter (Aim 2), thereby providing the molecular basis for substrate recognition, CPS translocation, as well as interaction with periplasmic and OM transporter components. To this end, we engineered a standard E. coli laboratory strain to produce a polysaccharide capsule from plasmid-encoded components. The expressed operon contains 9 genes and each can be removed from its expression plasmid by standard restriction enzyme digestion. Further, we also developed a molecular probe enabling the detection of the synthesized capsule on the cell surface, thereby correlating CPS production with the expression of the biosynthetic machinery. To integrate our functional analyses with a 3D structure of the CPS ABC transporter, we purified a stable transporter in complex with its periplasmic subunit that likely stabilizes interactions with the OM pore. We will use cryo electron microscopy to determine the transporter?s structure in different nucleotide-bound states. Combined, our proposed research will provide the molecular basis for CPS secretion and lay the foundation for structure-guided drug development.