Jeremiah G. Johnson

Role of MrkJ, a Phosphodiesterase, in Type 3 Fimbrial Expression and Biofilm Formation in Klebsiella pneumoniae

Klebsiella pneumoniae is an opportunistic pathogen that has been shown to adhere to human extracellular matrices using the type 3 fimbriae. Introduction of plasmids carrying genes known to alter intracellular cyclic-di-GMP pools in Vibrio parahaemolyticus revealed that these genes also altered type 3 fimbrial surface expression in K. pneumoniae. Immediately adjacent to the type 3 fimbrial gene cluster is a gene, mrkJ, that is related to a family of bacterial genes encoding phosphodiesterases. We identify here a role for MrkJ, a functional phosphodiesterase exhibiting homology to EAL domain-containing proteins, in controlling type 3 fimbria production and biofilm formation in K. pneumoniae. Deletion of mrkJ resulted in an increase in type 3 fimbria production and biofilm formation as a result of the accumulation of intracellular cyclic-di-GMP. This gene was shown to encode a functional phosphodiesterase via restoration of motility in a V. parahaemolyticus strain previously shown to accumulate cyclic-di-GMP and in vitro using phosphodiesterase activity assays. The effect of the mrkJ mutation on type 3 fimbrial expression was shown to be at the level of mrkA gene transcription by using quantitative reverse transcription-PCR. These results reveal a previously unknown role for cyclic-di-GMP in type 3 fimbrial production.

Peptidoglycan LD-Carboxypeptidase Pgp2 Influences Campylobacter jejuni Helical Cell Shape and Pathogenic Properties, and Provides the Substrate for the DL-Carboxypeptidase Pgp1

Background: C. jejuni helical shape is important to pathogenesis. Results: Deletion of pgp2 results in loss of C. jejuni helical shape and change in peptidoglycan structure and pathogenic properties. Conclusion: Pgp2 is a ld-carboxypeptidase cleaving peptidoglycan tetrapeptides to tripeptides. Significance: Characterization of enzymes involved in C. jejuni peptidoglycan and cell shape maintenance is crucial to the understanding of fundamental properties of this organism. Despite the importance of Campylobacter jejuni as a pathogen, little is known about the fundamental aspects of its peptidoglycan (PG) structure and factors modulating its helical morphology. A PG dl-carboxypeptidase Pgp1 essential for maintenance of C. jejuni helical shape was recently identified. Bioinformatic analysis revealed the CJJ81176_0915 gene product as co-occurring with Pgp1 in several organisms. Deletion of cjj81176_0915 (renamed pgp2) resulted in straight morphology, representing the second C. jejuni gene affecting cell shape. The PG structure of a Δpgp2 mutant showed an increase in tetrapeptide-containing muropeptides and a complete absence of tripeptides, consistent with ld-carboxypeptidase activity, which was confirmed biochemically. PG analysis of a Δpgp1Δpgp2 double mutant demonstrated that Pgp2 activity is required to generate the tripeptide substrate for Pgp1. Loss of pgp2 affected several pathogenic properties; the deletion strain was defective for motility in semisolid agar, biofilm formation, and fluorescence on calcofluor white. Δpgp2 PG also caused decreased stimulation of the human nucleotide-binding oligomerization domain 1 (Nod1) proinflammatory mediator in comparison with wild type, as expected from the reduction in muropeptide tripeptides (the primary Nod1 agonist) in the mutant; however, these changes did not alter the ability of the Δpgp2 mutant strain to survive within human epithelial cells or to elicit secretion of IL-8 from epithelial cells after infection. The pgp2 mutant also showed significantly reduced fitness in a chick colonization model. Collectively, these analyses enhance our understanding of C. jejuni PG maturation and help to clarify how PG structure and cell shape impact pathogenic attributes.

Type 3 Fimbriae and Biofilm Formation Are Regulated by the Transcriptional Regulators MrkHI in Klebsiella pneumoniae

Klebsiella pneumoniae is an opportunistic pathogen which frequently causes hospital-acquired urinary and respiratory tract infections. K. pneumoniae may establish these infections in vivo following adherence, using the type 3 fimbriae, to indwelling devices coated with extracellular matrix components. Using a colony immunoblot screen, we identified transposon insertion mutants which were deficient for type 3 fimbrial surface production. One of these mutants possessed a transposon insertion within a gene, designated mrkI, encoding a putative transcriptional regulator. A site-directed mutant of this gene was constructed and shown to be deficient for fimbrial surface expression under aerobic conditions. MrkI mutants have a significantly decreased ability to form biofilms on both abiotic and extracellular matrix-coated surfaces. This gene was found to be cotranscribed with a gene predicted to encode a PilZ domain-containing protein, designated MrkH. This protein was found to bind cyclic-di-GMP (c-di-GMP) and regulate type 3 fimbrial expression.

More than One Way To Control Hair Growth: Regulatory Mechanisms in Enterobacteria That Affect Fimbriae Assembled by the Chaperone/Usher Pathway

Many gram-negative enterobacteria produce surface-associated fimbriae that facilitate attachment and adherence to eucaryotic cells and tissues. These organelles are believed to play an important role during infection by enabling bacteria to colonize specific niches within their hosts. One class of these fimbriae is assembled using a periplasmic chaperone and membrane-associated scaffolding protein that has been referred to as an usher because of its function in fimbrial biogenesis. The presence of multiple types of fimbriae assembled by the chaperone/usher pathway can be found both within a single bacterial species and also among different genera. One way of controlling fimbrial assembly in these bacteria is at the genetic level by positively or negatively regulating fimbrial gene expression. This minireview considers the mechanisms that have been described to control fimbrial gene expression and uses specific examples to demonstrate both unique and shared properties of such regulatory mechanisms.

High-Throughput Sequencing of Campylobacter jejuni Insertion Mutant Libraries Reveals mapA as a Fitness Factor for Chicken Colonization

Campylobacter jejuni is a leading cause of gastrointestinal infections worldwide, due primarily to its ability to asymptomatically colonize the gastrointestinal tracts of agriculturally relevant animals, including chickens. Infection often occurs following consumption of meat that was contaminated by C. jejuni during harvest. Because of this, much interest lies in understanding the mechanisms that allow C. jejuni to colonize the chicken gastrointestinal tract. To address this, we generated a C. jejuni transposon mutant library that is amenable to insertion sequencing and introduced this mutant pool into day-of-hatch chicks. Following deep sequencing of C. jejuni mutants in the cecal outputs, several novel factors required for efficient colonization of the chicken gastrointestinal tract were identified, including the predicted outer membrane protein MapA. A mutant strain lacking mapA was constructed and found to be significantly reduced for chicken colonization in both competitive infections and monoinfections. Further, we found that mapA is required for in vitro competition with wild-type C. jejuni but is dispensable for growth in monoculture.

Current and Potential Treatments for Reducing Campylobacter Colonization in Animal Hosts and Disease in Humans

Campylobacter jejuni is the leading cause of bacteria-derived gastroenteritis worldwide. In the developed world, Campylobacter is usually acquired by consuming under-cooked poultry, while in the developing world it is often obtained through drinking contaminated water. Once consumed, the bacteria adhere to the intestinal epithelium or mucus layer, causing toxin-mediated inhibition of fluid reabsorption from the intestine and invasion-induced inflammation and diarrhea. Traditionally, severe or prolonged cases of campylobacteriosis have been treated with antibiotics; however, overuse of these antibiotics has led to the emergence of antibiotic-resistant strains. As the incidence of antibiotic resistance, emergence of post-infectious diseases, and economic burden associated with Campylobacter increases, it is becoming urgent that novel treatments are developed to reduce Campylobacter numbers in commercial poultry and campylobacteriosis in humans. The purpose of this review is to provide the current status of present and proposed treatments to combat Campylobacter infection in humans and colonization in animal reservoirs. These treatments include anti-Campylobacter compounds, probiotics, bacteriophage, vaccines, and anti-Campylobacter bacteriocins, all of which may be successful at reducing the incidence of campylobacteriosis in humans and/or colonization loads in poultry. In addition to reviewing treatments, we will also address several proposed targets that may be used in future development of novel anti-Campylobacter treatments.

Crystal structure of the MrkD1P receptor binding domain of Klebsiella pneumoniae and identification of the human collagen V binding interface

Klebsiella species are members of the family enterobacteriaceae, opportunistic pathogens that are among the eight most prevalent infectious agents in hospitals. Among other virulence factors in Klebsiella, type 3 pili exhibit a unique binding pattern in the human kidney via interaction of two MrkD adhesion variants 1C1 and 1P to type IV and/or V collagen. However, very little is known about the nature of this recognition. Here we present the crystal structure of the plasmid born MrkD1P receptor domain (MrkDrd). The structure reveals a jelly‐roll β‐barrel fold comprising 17 β‐strands very similar to the receptor domain of GafD, the tip adhesin from the F17 pilus that recognizes n‐acetyl‐d‐glucosamine (GlcNAc). Analysis of collagen V binding of different MrkD1P mutants revealed that two regions were responsible for its binding: a pocket, that aligns approximately with the GlcNAc binding pocket of GafD involving residues R105 and Y155, and a transversally oriented patch that spans strands β2a, β9b and β6 including residues V49, T52, V91, R102 and I136. Taken together, these data provide structural and functional insights on MrkD1P recognition of host cells, providing a tool for future development of rationally designed drugs with the prospect of blocking Klebsiella adhesion to collagen V.

Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni. Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro. The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target.

Narrow-Spectrum Inhibitors of Campylobacter jejuni Flagellar Expression and Growth

ABSTRACT Campylobacter jejuni is a major cause of food-borne illness due to its ability to reside within the gastrointestinal tracts of chickens. Multiple studies have identified the flagella of C. jejuni as a major determinant of chicken colonization. An inhibitor screen of approximately 147,000 small molecules was performed to identify compounds that are able to inhibit flagellar expression in a reporter strain of C. jejuni. Several compounds that modestly inhibited motility of wild-type C. jejuni in standard assays were identified, as were a number of small molecules that robustly inhibited C. jejuni growth, in vitro. Examination of similar bacterial screens found that many of these small molecules inhibited only the growth of C. jejuni. Follow-up assays demonstrated inhibition of other strains of C. jejuni and Campylobacter coli but no inhibition of the closely related Helicobacter pylori. The compounds were determined to be bacteriostatic and nontoxic to eukaryotic cells. Preliminary results from a day-of-hatch chick model of colonization suggest that at least one of the compounds demonstrates promise for reducing Campylobacter colonization loads in vivo, although further medicinal chemistry may be required to enhance bioavailability.

The PAS Domain-Containing Protein HeuR Regulates Heme Uptake in Campylobacter jejuni

ABSTRACT Campylobacter jejuni is a leading cause of bacterially derived gastroenteritis. A previous mutant screen demonstrated that the heme uptake system (Chu) is required for full colonization of the chicken gastrointestinal tract. Subsequent work identified a PAS domain-containing regulator, termed HeuR, as being required for chicken colonization. Here we confirm that both the heme uptake system and HeuR are required for full chicken gastrointestinal tract colonization, with the heuR mutant being particularly affected during competition with wild-type C. jejuni. Transcriptomic analysis identified the chu genes—and those encoding other iron uptake systems—as regulatory targets of HeuR. Purified HeuR bound the chuZA promoter region in electrophoretic mobility shift assays. Consistent with a role for HeuR in chu expression, heuR mutants were unable to efficiently use heme as a source of iron under iron-limiting conditions, and mutants exhibited decreased levels of cell-associated iron by mass spectrometry. Finally, we demonstrate that an heuR mutant of C. jejuni is resistant to hydrogen peroxide and that this resistance correlates to elevated levels of catalase activity. These results indicate that HeuR directly and positively regulates iron acquisition from heme and negatively impacts catalase activity by an as yet unidentified mechanism in C. jejuni. IMPORTANCE Annually, Campylobacter jejuni causes millions of gastrointestinal infections in the United States, due primarily to its ability to reside within the gastrointestinal tracts of poultry, where it can be released during processing and contaminate meat. In the developing world, humans are often infected by consuming contaminated water or by direct contact with livestock. Following consumption of contaminated food or water, humans develop disease that is characterized by mild to severe diarrhea. There is a need to understand both colonization of chickens, to make food safer, and colonization of humans, to better understand disease. Here we demonstrate that to efficiently colonize a host, C. jejuni requires iron from heme, which is regulated by the protein HeuR. Understanding how HeuR functions, we can develop ways to inhibit its function and reduce iron acquisition during colonization, potentially reducing C. jejuni in the avian host, which would make food safer, or limiting human colonization. Annually, Campylobacter jejuni causes millions of gastrointestinal infections in the United States, due primarily to its ability to reside within the gastrointestinal tracts of poultry, where it can be released during processing and contaminate meat. In the developing world, humans are often infected by consuming contaminated water or by direct contact with livestock. Following consumption of contaminated food or water, humans develop disease that is characterized by mild to severe diarrhea. There is a need to understand both colonization of chickens, to make food safer, and colonization of humans, to better understand disease. Here we demonstrate that to efficiently colonize a host, C. jejuni requires iron from heme, which is regulated by the protein HeuR. Understanding how HeuR functions, we can develop ways to inhibit its function and reduce iron acquisition during colonization, potentially reducing C. jejuni in the avian host, which would make food safer, or limiting human colonization.