182 P.B. WYRICK

Commentary by J. Pearce:

Reality check - - but how real should we get?

Pris Wyrick has, more than any other chlamy- diologist, pioneered the use of primary cultures and polarized cell systems in the study of chla- mydia-host cell interactions. She makes an important point. Chlamydiologists as a whole (including this contributor), in looking at the attachment and entry of this mucosal pathogen, have made far too little use of more realistic host cell systems.

That heparan sulphate may act as a chlamy- dial ligand for attachment is a complex story. The intriguing evidence from the GPIC study that pH increase can shift attachment to a non-heparan- dependent mechanism [15] may throw light on some of the variability. But Dr. Wyrick's data and interpretation have an appealing clarity and sim- plicity that emphasize the need to broaden out the host cell systems that we use.

Publications in the enteric invasion field indi- cate use of a much wider range of cell culture models. And here I draw on the experience of my colleague, John Stephen, who has devel- oped a novel organ culture system [16] to eval- uate salmonella invasion and which he has compared with simpler models. What is inter- est ing is to see how the d i f ferent models behave. For example, 3 virulent and 4 avirulent Salmonella typhimur ium strains whose inva- siveness had been assessed in vivo were tested in HEp-2 cells and in the rabbit ileum organ cul- ture. In HEp-2 cells the virulent strains were invasive and 3 of the 4 avirulent strains were hypoinvasive - - yet the other, SL1027, was highly invasive [17]. The mucosal organ culture of rabbit ileum correctly reproduced behaviour in vivo - - all the virulent strains were invasive and the avirulent strains non-invasive [16]. In other work, TnphoA mutants, which had been selected as hypoinvasive in HEp-2 cells, were as invasive as the parent strains in the organ cul- ture [18].

If HEp-2 cells are a poor model, what about others? S. typhimur ium and S. dublin strains showed similar invasiveness in bovine loops in vivo, but the S. dublin strains were significantly less invasive in either polarized MDCK cells or Int 407 cells [19]. And in other studies of the muco- sal organ culture an S. typhimurium invH mutant was weakly invasive in vivo but invasive in organ culture. This may have been because the culture system failed to produce mucus to trap the mutant in the manner seen in vivo (A.J. Bolton and J. Stephen, unpublished). Caco-2 cells, con- sidered the best polarized cell culture for model-

ling the small intestine, did not reflect the dam- age and destruction seen when an S. dubl in strain invaded the pig or calf intestine, because the Caco-2 cell tight junctions were not attacked (A.J. Bolton and J. Stephen, unpublished).

So what is the message from these observa- tions? Every artificial system is a compromise and polarized epithelial cultures have their limita- tions. The gut organ culture system [16] was designed to analyse invasion mutants. It probably could not be adapted for the study of conjunctival or genitourinary mucosa. Nor is it without its defects. But at the very least, chlamydiologists should consider comparison of HeLa, McCoy, etc. with a polarized cell system. This could provide useful insights in attachment-entry studies - - and maybe support or deny the relevance of the data gained in the simpler systems.

John Pearce Microbial Molecular Genetics School of Biological Sciences University of Birmingham Birmingham B15 2-1-1-, UK

Commentary by E. Kihlstr6m :

In the above letter Dr. Wyrick addresses the important issue of how we should design in vitro experimental systems for microorganism-host cell interactions to be able to extrapolate to in vivo events. As examples, she discusses adhesion of Chlamydia and Yersinia to eucaryotic cells.

The different biovars and serovars of Chla- mydia trachomatis give rise to various infections. Some serovars cause acute infections in the gen- ital tract, others lead to trachoma, and still others are more invasive, such as the lymphogranu- Ioma venereum (LGV) biovars. The molecular events culminating in these outcomes are not fully understood. I agree that the recent finding that heparan sulphate can act as an adhesin and is differently expressed in LGV and trachoma strains may be a reflection of biological differ- ences in vivo. Dr. Wyrick's approach to use polarized human genital epithelium as physio- logical target cells is certainly worth following.

However, it is not only the properties of the host cell that have to be controlled. Bacterial viru- lence factors that interact with and affect host cells are differently expressed under various cul- ture conditions. Production of capsule substance in group B streptococci is inhibited at pH 5, mak- ing the bacteria more susceptible to phagocyto- sis (Kihlstr6m et al. to be published). Anaerobic culture conditions of Salmonella bacteria pro- mote their adhesion to epithelial cells [20]. Thus,