We initiated this study to elucidate the mechanisms responsible for receptor-independent uptake of iron and heme from Hb, based on the previous isolation of gonococcal mutants that were able to grow on Hb despite mutational loss of HpuA, the lipoprotein coreceptor that normally is essential for the function of the HpuAB receptor (6
). The hgbX
mutants fell into two general classes based on differences in their susceptibilities to a variety of antimicrobial agents. Although all were more susceptible to generally hydrophobic antibiotics and TX-100 than the wild type, some were particularly susceptible. One of the two classes of mutants turned out to have a point mutation near the 3′ end of pilQ
), whereas the other was normal in all tested genes. Efforts to clone the gene(s) responsible for the remaining uncharacterized hgbX
mutants other than pilQ
by the same strategy used to isolate the pilQ1
mutation have been unsuccessful. The results described in this communication showed that the Hb+
phenotype of the pilQ1
mutants was neither dependent on HpuAB or TonB nor on Hb binding to whole cells. Rather, growth on Hb was due to the release of free heme from Hb and entry through a channel or channels that did not depend on the Hb receptor. We presume that heme was released from Hb on the cell surface or in the extracellular medium because HSA prevented the entry of heme. So far, there has not been any report of Hb entry in any bacterial species. The relatively large sizes of albumin and Hb (molecular weights of ca. 69,000 and 64,500, respectively) and the funnel-shaped cavity in the PilQ macromolecule (9
) make it unlikely that either could enter through the PilQ channel.
Demonstration that the pilQ1 mutation resulted in increased entry of heme, TX-100, and a variety of antibiotics was surprising, since PilQ is not known to facilitate the entry of exogenous compounds. It is likely that only a few mutations in pilQ result in this phenotype, since the same F563L mutation was isolated independently twice. Very recently, an additional and different pilQ mutation has been isolated that results in increased resistance to antibiotics, further suggesting that wild-type PilQ allows the entry of a variety of compounds (R. Nicholas et al., unpublished data). Our demonstration that the phenotype was very closely linked to pilQ1 and was lost by insertional inactivation of pilQ1 suggests that the effects were mediated by changes within PilQ. We cannot absolutely exclude the possibility that the PilQ F563L mutation provides these phenotypes by affecting membrane integrity or another secondary effect. The simplest hypothesis is that PilQ can directly allow entry of these molecules into the bacterial cell and that the F563L mutation increases the number of molecules that can efficiently utilize this route.
The conclusion that PilQ forms not only a pore for exit of the assembled pilus fibril (13
) but also a channel for entry of heme and various antimicrobial agents is strengthened by the additional observation that loss of pilT
function decreased the effects of the pilQ1
mutation. PilT is responsible for twitching motility mediated by retraction of the pilus fibril (27
). It also is necessary for degradation of the pilus fibril when PilQ is not available for export of the intracellular assembled pilus fibril; in the absence of both PilQ and PilT, cell viability is lost unless other secondary mutations in pilE
prevent formation of what would otherwise be toxic intracellular pilus fibrils (52
). PilT is also required for the transport of DNA into the cell for genetic transformation (48
). Our observation that inactivation of pilT
in strains carrying the pilQ1
missense mutation resulted in decreased sensitivity to heme, TX-100, and antibiotics argues that PilT normally increases the entry properties of the PilQ macromolecular complex. Perhaps the PilQ-dependent entry of small molecules shares mechanistic properties with pilus retraction, where the assembled pilus is imported back into the cell. It is not clear whether heme and antimicrobial agents bind to pili or to PilQ, but modestly increased sensitivity to heme and certain antimicrobial agents in a ΔpilE
construct (Fig. and Table ) suggests that either extruded pili compete with exogenous compounds for PilQ or pili partially impeded passage of these compounds through the PilQ channels.
Many bacteria possess receptors for binding and utilizing heme-carrying compounds (43
). The entrance of heme in gonococci is known to be TonB independent (3
), and no clear evidence for a heme receptor in the pathogenic Neisseria
spp. has been produced. Our results demonstrated that some heme enters through a PilT- and PilQ-dependent pathway, but there almost certainly must be an additional mechanism for heme entry as well, since strains lacking both an Hb receptor and PilQ were able to grow on free heme (Fig. ). It was curious that there was no toxicity when Hb was the source of heme, as opposed to adding free heme to the media. This may reflect relatively low concentrations of free heme released by Hb, compared to the amounts added to the wells in the assays used. Heme is known to be toxic for bacteria when in excess (40
). Indeed, use of iron regulated receptors for obtaining heme from Hb under physiological circumstances may help to control the amount of heme that enters the cell, thereby avoiding toxicity.
Recent structural studies have utilized electron microscopy to establish proposed quaternary structures for members of the secretin family. According to Collins et al. (8
), the assembled meningococcal PilQ product takes the form of a doughnut composed of 12 identical subunits, surrounding a central cavity. When viewed from the side, the assembly is proposed to have a rounded, conical profile. Viewed from the top, the open end of the central channel apparently is just large enough to accommodate the pilus fibril, and the tapered end presumably blocks the channel (9
). How the PilQ macromolecular assembly might be gated open so as to allow exit of pilus fibrils has been the subject of speculation involving the propulsive force of the pilus, which is sufficient to cause membrane protrusions in cells lacking PilQ and PilT (52
). Such a mechanism might account for the specificity of the channel for pili. The only other function associated with the pilus-PilQ channel has been uptake of transforming DNA, which depends on pili, PilC, PilT, and other members of the complex (51
). How DNA enters through this channel remains unclear; however, our data suggest that pilQ1
changes the specificity of the channel to allow increased entry of other molecules into the cell.
Studies on certain other members of the secretin family (34
) have demonstrated gated channel properties. The pIV secretin for the filamentous phage f1 forms an ion conductive pore in planar lipid bilayers, and mutant forms of the protein allow import of molecules in whole cells to which E. coli
normally is resistant (28
). Entry through the pIV channel was decreased by production of phage that could not be released from the cell surface (29
). The Klebsiella oxytoca
secretin PulD also forms ion conductive channels in planar lipid bilayers (31
). Each of these secretins has a quaternary shape similar to that proposed for PilQ (8
). Future studies on PilQ might be expected to show voltage-dependent ion conductive channels in planar lipid bilayers, and mutants of PilQ such as the one we describe should alter the properties of the channel. These mutants hopefully also will help us to understand how PilQ interacts with other members of the complex (PilT, PilP, and PilC) in the secretion of pili and the mechanisms and physiological roles for entry of exogenous compounds, including heme and antibiotics through PilQ.