System specificity of the TpsB transporters of coexpressed two-partner secretion systems of Neisseria meningitidis

Abstract

The two-partner secretion (TPS) systems of Gram-negative bacteria consist of a large secreted exoprotein (TpsA) and a transporter protein (TpsB) located in the outer membrane. TpsA targets TpsB for transport across the membrane via its ∼30-kDa TPS domain located at its N terminus, and this domain is also the minimal secretory unit. Neisseria meningitidis genomes encode up to five TpsAs and two TpsBs. Sequence alignments of TPS domains suggested that these are organized into three systems, while there are two TpsBs, which raised questions on their system specificity. We show here that the TpsB2 transporter of Neisseria meningitidis is able to secrete all types of TPS domains encoded in N. meningitidis and the related species Neisseria lactamica but not domains of Haemophilus influenzae and Pseudomonas aeruginosa. In contrast, the TpsB1 transporter seemed to be specific for its cognate N. meningitidis system and did not secrete the TPS domains of other meningococcal systems. However, TpsB1 did secrete the TPS2b domain of N. lactamica, which is related to the meningococcal TPS2 domains. Apparently, the secretion depends on specific sequences within the TPS domain rather than the overall TPS domain structure.

Fig 1

(A) Chromosomal organization of the TPS systems in N. meningitidis H44/76, as identified in the genome sequence (11, 12). The proteins encoded by the ORFs are indicated below. Red, system 1; blue, system 2; green, system 3. The dark lines indicate the parts of the tpsA genes used for the truncated TpsA ORFs, which comprise the signal peptide (sp) and the TPS domain. The white arrows between the ORFs encoding TpsA2a and TpsB2 are ORFs not encoding TPS proteins. The ORFs are believed to be part of an operon (5). (B) Graphical representation of the various meningococcal expression constructs used for this study. The constructs containing a truncated TpsA are given on the left, and the corresponding constructs of a truncated TpsA ORF in combination with a TpsB ORF are given on the right. Colors designate the different systems described for panel A.

Fig 2

Expression and secretion of TPS constructs in N. meningitidis HB-1, HB-1 tpsB1::kan, and HB-1 tpsB2::kan. (A) Immunoblots of cell lysates (C) and concentrated culture supernatants (S) of cells grown in the presence (+) or absence (−) of 1 mM IPTG to induce expression of the TPS construct. The bacterial strains grown are indicated on the top, while the TPS constructs with which the strains were transformed are indicated on the left. The blots were incubated with antisera against the TpsB1, TpsB2, TPS1, and TPS2 domains; the His tag was included in the TPS3 domain, as indicated on the right. *, the TPS1 domain secreted by the TpsB2 protein. (B) Immunoblot of samples of N. meningitidis HB-1 containing the TPS1 construct induced with different amounts of IPTG. The blot was incubated with antiserum against the TPS1 domain. The TPS1 bands are indicated by black arrowheads. conc. sup., concentrated culture supernatants. (C) Immunoblot of samples of N. meningitidis HB-1 containing the TPS1 construct induced or not with IPTG. The blot was incubated with antiserum against the TPS1 domain, focusing on the endogenous full-length TpsA1. The TpsA1 bands in the cell lysates of ∼240 kDa and ∼200 kDa (5) are indicated by black arrowheads. The open arrowhead indicates a background band recognized by the anti-TPS1 serum (5). *, the ∼240-kDa band accumulating in the cell fractions as a result of overproduction of the TPS1 domain. The positions of the M_w markers are indicated on the left in each panel.

Fig 3

Complementation of TPS constructs in N. meningitidis HB-1 tpsB1::kan tpsB2::gen. Immunoblots of cell lysates (C) and concentrated culture supernatants (S) of HB-1 tpsB1::kan tpsB2::gen containing TPS and TPS-TpsB constructs that were induced for expression by IPTG (+) or not (−), as indicated on the top. The blots were incubated with the antisera indicated on the right. (A) Immunoblot containing samples of cells expressing the TPS2 and TPS2-TpsB2 constructs incubated with antisera against the TpsB2 and TPS2 domains, as indicated on the right. To analyze the leakage of cellular content to the supernatant, we ultracentrifuged the supernatant and included samples of the high-speed-centrifugation supernatant (hsS) and high-speed-centrifugation pellet (hsP) on the blot. Black arrowheads, the detected TPS2b domain and TpsB2; lane M, a marker lane included in the blot. (B) Immunoblots containing samples of cells expressing the TPS1, TPS1-TpsB1, and TPS1-TpsB2 constructs incubated with antisera against the TpsB1, TpsB2, and TPS1 domains. Less of the supernatant of the TPS1-TpsB1 construct was loaded on the gel to prevent overexposure. (C) Immunoblots of HB-1 tpsB1::kan tpsB2::gen expressing the TPS3 and TPS3-TpsB2 constructs incubated with antisera against TpsB2 and the His tag included in the TPS3 domain. The positions of the M_w markers are indicated on the right in panel A and on the left in panels B and C.

Fig 4

Expression and secretion of N. lactamica TPS constructs in N. meningitidis HB-1, HB-1 tpsB1::kan tpsB2::gen, HB-1 tpsB1::kan, and HB-1 tpsB2::kan. Immunoblots of cell lysates (C) and culture supernatants (S) of cells grown in the presence (+) or absence (−) of IPTG to induce expression of the TPS construct. The bacterial strains are indicated on the top, while the TPS constructs with which the strains were transformed are indicated on the left. The blots were incubated with antisera against the His tag included in the TPS domains, as indicated on the right. The positions of the M_w markers are indicated on the left. *, the TPS-NL2b domain secreted by the meningococcal TpsB1 protein.

Fig 5

Structural models of the neisserial TPS domains. (Top) Cartoon representations of the tree TPS domain structures that have been solved to date (9, 39, 40), with their Protein Data Bank accession numbers given in parentheses; (middle and bottom) cartoon representations of structural models for the neisserial TPS domains that were compiled using Phyre (23). They show a large overall similarity, corroborating the structure-based alignments (see Fig. S4 in the supplemental material). The colored regions (red in TPS1; dark blue in TPS2a, TPS2b, and TPS-NL2b; light blue in TPS3 and TPS-NL4) indicate the regions of low confidence in the alignments. The cartoons were generated using Pymol.