Anthony T. Maurelli

A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis

Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia harbour genes for PG biosynthesis and exhibit susceptibility to ‘anti-PG’ antibiotics, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’). We used a novel approach to metabolically label chlamydial PG using d-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis were labelled with these probes throughout their biphasic developmental life cycle, and the results of differential probe incorporation experiments conducted in the presence of ampicillin are consistent with the presence of chlamydial PG-modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence so far that chlamydial species possess functional PG.

l,l-diaminopimelate aminotransferase, a trans-kingdom enzyme shared by Chlamydia and plants for synthesis of diaminopimelate/lysine

The synthesis of meso-diaminopimelic acid (m-DAP) in bacteria is essential for both peptidoglycan and lysine biosynthesis. From genome sequencing data, it was unclear how bacteria of the Chlamydiales order would synthesize m-DAP in the absence of dapD, dapC, and dapE, which are missing from the genome. Here, we assessed the biochemical capacity of Chlamydia trachomatis serovar L2 to synthesize m-DAP. Expression of the chlamydial asd, dapB, and dapF genes in the respective Escherichia coli m-DAP auxotrophic mutants restored the mutants to DAP prototrophy. Screening of a C. trachomatis genomic library in an E. coli ΔdapD DAP auxotroph identified ct390 as encoding an enzyme that restored growth to the Escherichia coli mutant. ct390 also was able to complement an E. coli ΔdapD ΔdapE, but not a ΔdapD ΔdapF mutant, providing genetic evidence that it encodes an aminotransferase that may directly convert tetrahydrodipicolinate to l,l-diaminopimelic acid. This hypothesis was supported by in vitro kinetic analysis of the CT390 protein and the fact that similar properties were demonstrated for the Protochlamydia amoebophila homologue, PC0685. In vivo, the C. trachomatis m-DAP synthesis genes are expressed as early as 8 h after infection. An aminotransferase activity analogous to CT390 recently has been characterized in plants and cyanobacteria. This previously undescribed pathway for m-DAP synthesis supports an evolutionary relationship among the chlamydiae, cyanobacteria, and plants and strengthens the argument that chlamydiae synthesize a cell wall despite the inability of efforts to date to detect peptidoglycan in these organisms.

Staying alive: bacterial inhibition of apoptosis during infection

The ability of bacterial pathogens to inhibit apoptosis in eukaryotic cells during infection is an emerging theme in the study of bacterial pathogenesis. Prevention of apoptosis provides a survival advantage because it enables the bacteria to replicate inside host cells. Bacterial pathogens have evolved several ways to prevent apoptosis by protecting the mitochondria and preventing cytochrome c release, by activating cell survival pathways, or by preventing caspase activation. This review summarizes the most recent work on bacterial anti-apoptotic strategies and suggests new research that is necessary to advance the field.

In Vitro and In Vivo Functional Activity of Chlamydia MurA, a UDP-N-Acetylglucosamine Enolpyruvyl Transferase Involved in Peptidoglycan Synthesis and Fosfomycin Resistance

Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal DeltamurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.

Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1Tyr)

virR is the central regulatory locus required for coordinate temperature‐regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI‐Accl DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique Hpal restriction site resulted in a loss of VirR+ activity. The vir R ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone‐like protein, H‐NS. Based on the predicted amino acid sequence of E. coli H‐NS, only a single conservative base‐pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNATyr. These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.

Shigella flexneri LuxS Quorum-Sensing System Modulates virB Expression but Is Not Essential for Virulence

ABSTRACT Quorum-sensing systems regulate the expression of virulence factors in a wide variety of plant and animal pathogens, including members of the Enterobacteriaceae. Studies of Shigellavirulence gene expression have demonstrated that maximal expression of genes encoding the type III secretion system and its substrates and maximal activity of this virulence organelle occur at high cell density. In these studies, we demonstrate that the expression ofipa, mxi, and spa invasion operons is maximal in stationary-phase bacteria and that conditioned media derived from stationary-phase cultures enhance the expression of these loci. In contrast, expression of virB, a transcription factor essential for the expression of invasion loci, peaks in late log phase; accordingly, virB expression is enhanced by a signal(s) present in conditioned media derived from late-log-phase cultures. Autoinducer 2 (AI-2), a quorum signaling molecule active in late log phase, was synthesized by Shigella species and enteroinvasive Escherichia coli and shown to be responsible for the observed peak of virB expression. However, AI-2 does not influence invasion operon expression and is not required forShigella virulence, as mutants deficient in AI-2 synthesis are fully virulent. The implications of these findings with regard to both virB and invasion operon expression and the evolution of circuitries governing virulence gene expression are discussed.

Antivirulence Genes: Insights into Pathogen Evolution through Gene Loss

ABSTRACT The emergence of new pathogens and the exploitation of novel pathogenic niches by bacteria typically require the horizontal transfer of virulence factors and subsequent adaptation—a “fine-tuning” process—for the successful incorporation of these factors into the microbes genome. The function of newly acquired virulence factors may be hindered by the expression of genes already present in the bacterium. Occasionally, certain genes must be inactivated or deleted for full expression of the pathogen phenotype to occur. These genes are known as antivirulence genes (AVGs). Originally identified in Shigella, AVGs have improved our understanding of pathogen evolution and provided a novel approach to drug and vaccine development. In this review, we revisit the AVG definition and update the list of known AVGs, which now includes genes from pathogens such as Salmonella, Yersinia pestis, and the virulent Francisella tularensis subspecies. AVGs encompass a wide variety of different roles within the microbe, including genes involved in metabolism, biofilm synthesis, lipopolysaccharide modification, and host vasoconstriction. More recently, the use of one of these AVGs (lpxL) as a potential vaccine candidate highlights the practical application of studying AVG inactivation in microbial pathogens.

A system for identifying post‐invasion functions of invasion genes: requirements for the Mxi–Spa type III secretion pathway of Shigella flexneri in intercellular dissemination

Invasion and intercellular spread are hallmarks of Shigella pathogenicity. Invasion of the eukaryotic cell cytosol requires a type III secretion system (Mxi–Spa) and its cognate set of secreted Ipa invasins. Once intracellular, the IcsA protein directs a form of actin‐based motility that helps to drive intracellular bacterial movement, formation of cellular protrusions and cell‐to‐cell spread. Work in our laboratory has focused on identifying additional factors required for this intercellular form of dissemination. In this study, we sought to identify novel contributions of the type III secretion pathway to post‐invasion‐specific processes, distinct from its previously characterized roles in invasion. Studies of post‐invasion Ipa and Mxi–Spa functions are complicated by an absolute requirement for these virulence proteins in invasion. To circumvent this problem, we developed a system called TIER (for test of intracellular expression requirements), whereby specific ipa, mxi or spa loci are transiently expressed before infection of tissue culture cell monolayers (thus supporting invasion), but then repressed after invasion in the intracellular environment. Such invasive type III secretion mutants (called TIER mutants) were severely restricted in their ability to spread intercellularly and form plaques in confluent tissue culture cell monolayers. Intercellular spread defects were associated with the repression of most type III pathway components examined, including structural (MxiM and Spa33), secreted effector (IpaB, IpaC and IpaD) and regulatory elements (VirF and VirB). A kinetic analysis of bacterial growth in L2 cell monolayers showed that each of the TIER mutants was defective with respect to long‐term intracellular proliferation and viability. Examination of TIER mutant‐infected monolayers by electron microscopy revealed that the type III pathway was required for a late step in intercellular spread — bacterial escape from protrusion‐derived, double‐membrane‐bound vacuoles. The TIER mutants were eventually degraded in a process involving vacuolar acidification. Based on these findings, we propose that Ipa secretion via Mxi–Spa is required in the protrusion vacuole for double‐membrane lysis.

Transformation and isolation of allelic exchange mutants of Chlamydia psittaci using recombinant DNA introduced by electroporation.

To facilitate genetic investigations in the obligate intracellular pathogens Chlamydia, the ability to construct variants by homologous recombination was investigated in C. psittaci 6BC. The single rRNA operon was targeted with a synthetic 16S rRNA allele, harboring three nucleotide substitutions over 398 bp, which imparts resistance to kasugamycin (Ksm) and spectinomycin (Spc) and causes loss of one HpaI restriction site. A fourth, silent mutation was introduced 654 bp downstream in the beginning of the 23S rRNA gene. C. psittaci 6BC infectious particles were electroporated with various concentrations of circular or linearized plasmids containing different lengths of the rRNA region homologous to the chromosomal copy except for the four nucleotide substitutions. Ksm and Spc were added 18 h after inoculation onto confluent cell monolayers in the plaque assay. Resistant plaques were picked and expanded with selection 10 days later before collecting DNA for analysis by PCR, restriction mapping, sequencing, or Southern. Spontaneous resistance to Ksm and Spc was never observed in mock electroporated bacteria (frequency <6.2 × 10−9). Conversely, double resistance and replacement of the 16S rRNA gene were observed when C. psittaci was electroporated with the recombination substrates. Highest efficiency was obtained with 10 μg of circular vector prepared in a DNA methylase-deficient Escherichia coli (1.9 ± 1.1 × 10−6, n = 7). Coinheritance of the silent 23S rRNA mutation was seen in 46 of 67 recombinants analyzed, illustrating DNA exchange of up to 1,052 bp in length. These findings provide the first step toward genetic manipulation of Chlamydia.

MxiM and MxiJ, Base Elements of the Mxi-Spa Type III Secretion System of Shigella, Interact with and Stabilize the MxiD Secretin in the Cell Envelope

The type III secretion pathway is broadly distributed across many parasitic bacterial genera and serves as a mechanism for delivering effector proteins to eukaryotic cell surface and cytosolic targets. While the effectors, as well as the host responses elicited, differ among type III systems, they all utilize a conserved set of 9 to 11 proteins that together form a bacterial envelope-associated secretory organelle or needle complex. The general structure of the needle complex consists of a transenvelope base containing at least three ring-forming proteins (MxiD, MxiJ, and MxiG in Shigella) that is connected to a hollow needle-like extension that projects away from the cell surface. Several studies have shown that the initial steps in needle complex assembly require interactions among the base proteins, although specific details of this process remain unknown. Here we identify a role for another base element in Shigella, MxiM, in interactions with the major outer-membrane-associated ring-forming protein, MxiD. MxiM affects several features of MxiD, including its stability, envelope association, and assembly into homomultimeric structures. Interestingly, many of the effects were also elicited by the inner-membrane-associated base element, MxiJ. We confirmed that MxiM-MxiD and MxiJ-MxiD interactions occur in vivo in the cell envelope, and we present evidence that together these base elements can form a transmembrane structure which is likely an important intermediary in the process of needle complex assembly.