Microbiological Control for Affinity Capture Chromatography Processing: An Industry Perspective.

The purpose of this paper is to provide a summary of a BPOG-led industry survey of the microbiological control aspects of affinity chromatography processing in the biopharmaceutical industry. The document provides a summary of historical microbiological control concerns, coupled with industry-derived best practices, for material, equipment, and storage controls required to mitigate the potential for microbial ingress and contamination of chromatography resin and equipment. These best practice guidelines, which are derived from the members of the BPOG Bioburden Working Group, are intended to assist biopharmaceutical manufacturers to enhance microbial control and monitoring strategies for chromatography systems.

Regulation of virulence gene transcripts by the Francisella novicida orphan response regulator PmrA: role of phosphorylation and evidence of MglA/SspA interaction.

Francisella tularensis subsp. tularensis is the etiologic agent of tularemia and has been designated a category A biothreat agent by the CDC. Tularemia is characterized by replication and dissemination within host phagocytes. Intramacrophage growth is dependent upon the regulation of Francisella pathogenicity island (FPI) virulence genes, which is poorly understood. Two-component regulatory systems (TCS) are widely employed by Gram-negative bacteria to monitor and respond to environmental signals. Virulent strains of F. tularensis subsp. tularensis are devoid of classical, tandemly arranged TCS genes, but orphaned members, such as that encoding the response regulator PmrA, have been identified. In the F. novicida model system, previous work has shown that a pmrA mutant shows decreased expression of FPI genes, is deficient for intramacrophage growth, and is avirulent in the mouse model. Here, we determine that phosphorylation aids PmrA binding to regulated promoters pmrA and the FPI-encoded pdpD, and KdpD is the histidine kinase primarily responsible for phosphorylation of PmrA at the aspartic acid at position 51 (D51). A strain expressing PmrA D51A retains some DNA binding but exhibits reduced expression of the PmrA regulon, is deficient for intramacrophage replication, and is attenuated in the mouse model. With regard to virulence gene induction, PmrA coprecipitates with the FPI transcription factors MglA and SspA, which bind RNA polymerase. Together, these data suggest a model of Francisella gene regulation that includes a TCS consisting of KdpD and PmrA. Once phosphorylated, PmrA binds to regulated gene promoters recruiting free or RNA polymerase-bound MglA and SspA to initiate FPI gene transcription.

Identification of an orphan response regulator required for the virulence of Francisella spp. and transcription of pathogenicity island genes.

Francisella tularensis is a category A agent of biowarfare/biodefense. Little is known about the regulation of virulence gene expression in Francisella spp. Comparatively few regulatory factors exist in Francisella, including those belonging to two-component systems (TCS). However, orphan members of typical TCS can be identified. To determine if orphan TCS members affect Francisella gene expression, a gene encoding a product with high similarity to the Salmonella PmrA response regulator (FTT1557c/FNU0663.2) was deleted in Francisella novicida (a model organism for F. tularensis). The F. novicida pmrA mutant was defective in survival/growth within human and murine macrophage cell lines and was 100% defective in virulence in mice at a dose of up to 10(8) CFU. In addition, the mutant strain demonstrated increased susceptibility to antimicrobial peptide killing, but no differences were observed between the lipid A of the mutant and the parental strain, as has been observed with pmrA mutants of other microbes. The F. novicida pmrA mutant was 100% protective as a single-dose vaccine when challenge was with 10(6) CFU of F. novicida but did not protect against type A Schu S4 wild-type challenge. DNA microarray analysis identified 65 genes regulated by PmrA. The majority of these genes were located in the region surrounding pmrA or within the Francisella pathogenicity island (FPI). These FPI genes are also regulated by MglA, but MglA does not regulate pmrA, nor does PmrA regulate MglA. Thus, the orphan response regulator PmrA is an important factor in controlling virulence in F. novicida, and a pmrA mutant strain is an effective vaccine against homologous challenge.