Ifor R. Beacham

A Type IV Pilin, PilA, Contributes to Adherence of Burkholderia pseudomallei and Virulence In Vivo

ABSTRACT The Burkholderia pseudomallei K96243 genome contains multiple type IV pilin-associated loci, including one encoding a putative pilus structural protein (pilA). A pilA deletion mutant has reduced adherence to human epithelial cells and is less virulent in the nematode model of virulence and the murine model of melioidosis, suggesting a role for type IV pili in B. pseudomallei virulence.

A genomic survey of positive selection in Burkholderia pseudomallei provides insights into the evolution of accidental virulence

Certain environmental microorganisms can cause severe human infections, even in the absence of an obvious requirement for transition through an animal host for replication (“accidental virulence”). To understand this process, we compared eleven isolate genomes of Burkholderia pseudomallei (Bp), a tropical soil microbe and causative agent of the human and animal disease melioidosis. We found evidence for the existence of several new genes in the Bp reference genome, identifying 282 novel genes supported by at least two independent lines of supporting evidence (mRNA transcripts, database homologs, and presence of ribosomal binding sites) and 81 novel genes supported by all three lines. Within the Bp core genome, 211 genes exhibited significant levels of positive selection (4.5%), distributed across many cellular pathways including carbohydrate and secondary metabolism. Functional experiments revealed that certain positively selected genes might enhance mammalian virulence by interacting with host cellular pathways or utilizing host nutrients. Evolutionary modifications improving Bp environmental fitness may thus have indirectly facilitated the ability of Bp to colonize and survive in mammalian hosts. These findings improve our understanding of the pathogenesis of melioidosis, and establish Bp as a model system for studying the genetics of accidental virulence.

Transcriptional co‐activation at the ansB promoters: involvement of the activating regions of CRP and FNR when bound in tandem

Previous work with semi‐synthetic promoters containing a single CRP binding site centred at 41.5 bp from the transcription start site has demonstrated enhanced transcription (synergism) when a second binding site, for CRP or FNR, is placed upstream at around −91 bp. The ansB promoter in Escherichia coli is co‐activated in a co‐dependent manner by one dimer each of CRP and FNR protein whose binding sites are at around −91 and −41 bp, respectively, from the transcription start site. Similarly, the homologous ansB promoter in Salmonella is co‐activated by two dimers of CRP which function synergistically. The binding sites at the E. coli promoter have been changed by mutation to provide a number of active promoter derivatives carrying other combinations of FNR and CRP binding sites. The co‐dependent versus synergistic interaction of these activators and their requirement for known activating regions have been examined. The results demonstrate that FNR can co‐activate when located upstream at around −91 bp in combination with either FNR or CRP downstream. When FNR occupies the downstream site the promoter is co‐dependent on an upstream activator, but not when CRP occupies this site. Activating region 1 in CRP (defined by substitutions at residue H159) and its putative equivalent in FNR (defined by substitutions at S73) are mainly required in the upstream activator; the putative equivalent in FNR of activating region 3 of CRP (defined by substitutions at G85 and K52, respectively) is mainly required in the dimer which binds downstream. Activating region 1 of FNR is required only in the downstream subunit of the upstream activator in a promoter which is co‐dependent on two FNR dimers. These data suggest that both bound upstream and downstream activators interact with RNA polymerase to promote transcription, and that co‐dependence is determined by the nature of the activator plus the promoter context.

Nasal‐Associated Lymphoid Tissue and Olfactory Epithelium as Portals of Entry for Burkholderia pseudomallei in Murine Melioidosis

BACKGROUND Burkholderia pseudomallei, the causative agent of melioidosis, is generally considered to be acquired via inhalation of dust or water droplets from the environment. In this study, we show that infection of the nasal mucosa is potentially an important portal of entry in melioidosis. METHODS After intranasal inoculation of mice, infection was monitored by bioluminescence imaging and by immunohistological analysis of coronal sections. The bacterial loads in organ and tissue specimens were also monitored. RESULTS Bioluminescence imaging showed colonization and replication in the nasal cavity, including the nasal-associated lymphoid tissue (NALT). Analysis of coronal sections and immunofluorescence microscopy further demonstrated the presence of infection in the respiratory epithelium and the olfactory epithelium (including associated nerve bundles), as well as in the NALT. Of significance, the olfactory epithelium and the brain were rapidly infected before bacteria were detected in blood, and a capsule-deficient mutant infected the brain without significantly infecting blood. CONCLUSIONS These data suggest that the olfactory nerve is the route of entry into the brain and that this route of entry may be paralleled in cases of human neurologic melioidosis. This study focuses attention on the upper respiratory tract as a portal of entry, specifically focusing on NALT as a route for the development of systemic infection via the bloodstream and on the olfactory epithelium as a direct route to the brain.

Pathogens Penetrating the Central Nervous System: Infection Pathways and the Cellular and Molecular Mechanisms of Invasion

SUMMARY The brain is well protected against microbial invasion by cellular barriers, such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). In addition, cells within the central nervous system (CNS) are capable of producing an immune response against invading pathogens. Nonetheless, a range of pathogenic microbes make their way to the CNS, and the resulting infections can cause significant morbidity and mortality. Bacteria, amoebae, fungi, and viruses are capable of CNS invasion, with the latter using axonal transport as a common route of infection. In this review, we compare the mechanisms by which bacterial pathogens reach the CNS and infect the brain. In particular, we focus on recent data regarding mechanisms of bacterial translocation from the nasal mucosa to the brain, which represents a little explored pathway of bacterial invasion but has been proposed as being particularly important in explaining how infection with Burkholderia pseudomallei can result in melioidosis encephalomyelitis.

Burkholderia pseudomallei Penetrates the Brain via Destruction of the Olfactory and Trigeminal Nerves: Implications for the Pathogenesis of Neurological Melioidosis

ABSTRACT Melioidosis is a potentially fatal disease that is endemic to tropical northern Australia and Southeast Asia, with a mortality rate of 14 to 50%. The bacterium Burkholderia pseudomallei is the causative agent which infects numerous parts of the human body, including the brain, which results in the neurological manifestation of melioidosis. The olfactory nerve constitutes a direct conduit from the nasal cavity into the brain, and we have previously reported that B. pseudomallei can colonize this nerve in mice. We have now investigated in detail the mechanism by which the bacteria penetrate the olfactory and trigeminal nerves within the nasal cavity and infect the brain. We found that the olfactory epithelium responded to intranasal B. pseudomallei infection by widespread crenellation followed by disintegration of the neuronal layer to expose the underlying basal layer, which the bacteria then colonized. With the loss of the neuronal cell bodies, olfactory axons also degenerated, and the bacteria then migrated through the now-open conduit of the olfactory nerves. Using immunohistochemistry, we demonstrated that B. pseudomallei migrated through the cribriform plate via the olfactory nerves to enter the outer layer of the olfactory bulb in the brain within 24 h. We also found that the bacteria colonized the thin respiratory epithelium in the nasal cavity and then rapidly migrated along the underlying trigeminal nerve to penetrate the cranial cavity. These results demonstrate that B. pseudomallei invasion of the nerves of the nasal cavity leads to direct infection of the brain and bypasses the blood-brain barrier. IMPORTANCE Melioidosis is a potentially fatal tropical disease that is endemic to northern Australia and Southeast Asia. It is caused by the bacterium Burkholderia pseudomallei, which can infect many organs of the body, including the brain, and results in neurological symptoms. The pathway by which the bacteria can penetrate the brain is unknown, and we have investigated the ability of the bacteria to migrate along nerves that innervate the nasal cavity and enter the frontal region of the brain by using a mouse model of infection. By generating a mutant strain of B. pseudomallei which is unable to survive in the blood, we show that the bacteria rapidly penetrate the cranial cavity using the olfactory (smell) nerve and the trigeminal (sensory) nerve that line the nasal cavity. Melioidosis is a potentially fatal tropical disease that is endemic to northern Australia and Southeast Asia. It is caused by the bacterium Burkholderia pseudomallei, which can infect many organs of the body, including the brain, and results in neurological symptoms. The pathway by which the bacteria can penetrate the brain is unknown, and we have investigated the ability of the bacteria to migrate along nerves that innervate the nasal cavity and enter the frontal region of the brain by using a mouse model of infection. By generating a mutant strain of B. pseudomallei which is unable to survive in the blood, we show that the bacteria rapidly penetrate the cranial cavity using the olfactory (smell) nerve and the trigeminal (sensory) nerve that line the nasal cavity.

Temperature regulation of protease in Pseudomonas fluorescens LS107d2 by an ECF sigma factor and a transmembrane activator.

The production of extracellular enzymes by Pseudomonas fluorescens is important with respect to phytopathogenesis and, in the case of psychrotrophic strains, food spoilage. The production of extracellular protease has been previously reported to be dependent on temperature in psychrotrophic strains of P. fluorescens; production is decreased above the optimum growth temperature with a relatively small change in growth rate. In this work, a transposon mutant of P. fluorescens LS107d2 has been isolated which, in contrast to the wild-type strain, is completely protease deficient at 29 degrees C, above the optimum growth temperature of 25 degrees C, but which produces protease at 23 degrees C. Further analysis revealed that this mutation is in a gene (prtR) which is part of a dicistronic operon, prtIR, in which the two genes are translationally coupled. Evidence is presented that prtI encodes a sigma factor related to others involved in extracytoplasmic functions (ECF sigma factors) and that prtR encodes a novel transmembrane activator of PrtI. PrtI, like PrtR, is also required for protease production at 29 degrees C but not at 23 degrees C. Analysis of the amino acid sequence of PrtR indicates that it is functionally related to a group of membrane-associated anti-sigma factors and a few transmembrane regulators, but is not significantly sequence related. Complementation analysis indicates that PrtR may also interact with sigma factors other than PrtI. The promoter region of the protease-encoding gene (aprX) in LS107d2 has been identified and has sequence features which could indicate interaction with either an ECF sigma factor or a primary sigma factor.

Co‐dependent positive regulation of the ansBF promoter of Escherichia coli by CRP and the FNR protein: a molecular analysis

Transcription of the ansB gene, encoding l‐asparaginase II, is positively regulated by cAMP receptor protein (CRP) and by the product of the fnr gene, the FNR protein. These global regulatory proteins mediate the expression of ansB in Escherichia coli in response to carbon source and to anaerobiosis, respectively, and are required concurrently for optimal ansB expression. The mechanism whereby CRP and FNR interact co‐operatively with the ansB promoter to achieve transcription has not previously been established. We have utilized an ansB‘‐’1lacZ fusion, in conjunction with deletion analysis and site‐directed mutagenesis, to identify two sites which interact with these regulatory proteins In the ansB promoter. The first is an FNR site, centred 41.5 bp upstream of the major transcriptional start site. The second site, located 28 bp upstream of the FNR site, is the site of CRP regulation. This site is homologous to both the CRP and FNR binding‐site consensus sequences and may respond to both CRP and FNR. The concurrent requirement for CRP and FNR for optimal expression of ansB may be explained if, first, essentially no transcription occurs unless the FNR is bound at the downstream site, and, second, the level of transcription when FNR alone is present is enhanced when CRP binds at the upstream site.

Rare codons in E. coli and S. typhimurium signal sequences

Codon usage has been examined in the signal sequences of 27 genes encoding proteins which possess leader peptides, and are inner‐membrane located or exported. The results have been compared with codon usage in the corresponding coding sequences of most of the mature proteins. A bias is observed in the usage of rare codons for two of the three hydrophobic amino acids for which there are rare codons. Since hydrophobic residues are predominant in leader peptides, we suggest that a resulting concentration of rare codons in the signal sequence may play a role (or have played a role in the evolutionary past) in the secretion process by delaying translation.

Biased codon usage in signal peptides : a role in protein export

The signal peptide of proteins exported via the general secretory pathway encodes structural features that enable the targeting and export of the protein to the periplasm. Recent studies have shown biased codon usage at the second amino acid position and a high usage of non-optimal codons within the signal peptide. Altering these biases in codon usage can have deleterious effects on protein folding and export. We propose that these codon-usage biases act in concert to optimize the export process through modulating ribosome spacing on the transcript. This highlights a new aspect of protein export and implies that codon usage in the signal peptide encodes signals that are important for protein targeting and export to the periplasm.