The Secretion of Toxins and Other Exoproteins of Cronobacter: Role in Virulence, Adaption, and Persistence

Abstract

: Cronobacter species are considered an opportunistic group of foodborne pathogenic bacteria capable of causing both intestinal and systemic human disease. This review describes common virulence themes shared among the seven Cronobacter species and describes multiple exoproteins secreted by Cronobacter, many of which are bacterial toxins that may play a role in human disease. The review will particularly concentrate on the virulence factors secreted by C. sakazakii, C. malonaticus, and C. turicensis, which are the primary human pathogens of interest. It has been discovered that various species-specific virulence factors adversely affect a wide range of eukaryotic cell processes including protein synthesis, cell division, and ion secretion. Many of these factors are toxins which have been shown to also modulate the host immune response. These factors are encoded on a variety of mobile genetic elements such as plasmids and transposons; this genomic plasticity implies ongoing re-assortment of virulence factor genes which has complicated our efforts to categorize Cronobacter into sharply defined genomic pathotypes.

Keywords: adherence factors; efflux pumps; iron transport; osmotic stress response; outer membrane proteins; plasmids; protein secretion systems; quorum sensing systems; virulence factors.

Figure 1

Sequence alignment of pESA3, pCS2, pSP291–1 and pCTU1 produced on the CGView Server from the Stothard Research Group [55] that uses BLAST analysis to illustrate conserved and missing genomic sequences (Available online: http://stothard.afns.ualberta.ca/cgview_server/; last accessed 12/20/2019). Two circular plasmid genomes, pCUNV1 (NZ_CP012258) and pCTU1 (NC_013283), were compared against the reference pESA3 (NC_009780). GenBank annotations of the reference pESA3 (CDS in blue arranged in two outside rings) were downloaded as a GFF file for analysis using the default configuration on the CGView server. Select genes or loci of interest are shown as across the circular genomes as follows: Siderophore loci with Cronobactin gene, Iron ABC transporter genes, Type 6 Secretion System (T6SS), parAB genes and the toxin cpa gene are adapted from Franco et al. [47]. Missing regions identified by the BLAST analysis on the CGView server are shown as ‘gaps’ on each of the two circular genomes. Genes and loci missing in pCUNV1 or pCTU1 plasmids are in red. As expected, T6SS is seen only on the reference pESA3 from C. sakazakii while the toxin encoding cpa gene is absent in the plasmid pCTU1 from C. turicensis. Figure was adapted from Jang et al. [56].

Figure 2

Phylogenetic tree of the homologs of omptin, Cpa. The NCBI accession numbers of the proteins sequences used in the figure are as follows: Yersinia pestis, Pla (plasminogen activator, NP_857784); S. enterica Typhimirium, PgtE (outer membrane serine protease, AAF85951); Erwinia, PlaA (plasmid, NP_857613); C. sakazakii BAA-894, Cpa (plasmid, ESA_pESA3p05434); C. universalis NCTC 9529, Cpa (omptin family outer membrane protease, WP_007705717); E. coli, OmpT (outer membrane protease VII, AP_001210); E. coli, OmpP (outer membrane protease P, X74278); and Shigella flexneri, SopA (outer membrane protease, NP_858404). Forty-one amino acids were added to C. sakazakii Cpa protein in its N-terminal to correct the incomplete annotation of the protein in the GenBank record. The MUSCLE algorithm of the MEGA7 suite was used for multiple sequence alignment. Phylogenetic analyses were conducted in MEGA7 using the Maximum-Likelihood algorithm [77]. Three hundred nine amino acid positions across the protein were used for determining the distance between the homologs in the tree. Bar marker represents 0.1 amino acid differences. Confidence values given in the nodes were derived out of bootstrap test consisting of 500 iterations.

Figure 3

Mechanisms of plasminogen activation by C. sakazakii and its role in bacterial virulence. It is thought that a complex with plasminogen is formed when Cronobacter plasminogen activator (Cpa) is expressed by invasive C. sakazakii (cells invading a host’s circulatory system), which causes proteolysis and conversion of host plasminogen to plasmin. Plasmin bound (conjecture) to bacterial cell surfaces then catalyzes the degradation of fibrin polymers (fibrinolysis) which are major components of fibrin clots and the extracellular matrix. Additionally, Cpa can also inactivate α2-anti-plasmin which normally would break down plasmin. Thus, there is an unlimited activation of plasmin leading to increased fibrinolysis which in turns allows for increased invasiveness.